Multispecific antigen binding proteins and methods of use thereof

ABSTRACT

Disclosed herein are multispecific, such as bispecific, antigen binding proteins comprising a first antigen binding domain comprising a heavy chain variable domain and a light chain variable domain, and a second antigen binding domain comprising a single-domain antibody. Pharmaceutical compositions comprising the multispecific antigen binding proteins, kits and methods of use thereof are further provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority benefit of International PatentApplication No. PCT/CN2016/090703 filed Jul. 20, 2016, the contents ofwhich are incorporated herein by reference in their entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 761422000241SEQLIST.txt,date recorded: Jul. 10, 2017, size: 14 KB).

FIELD OF THE INVENTION

The present invention relates to multispecific antigen binding proteins(MABPs) comprising at least one single-domain antibody and methods ofuse thereof.

BACKGROUND OF THE INVENTION

Monoclonal antibodies (mAbs) have been widely used as therapeutic agentsto treat a variety of human diseases, such as cancer and autoimmunediseases. Currently, there are more than 30 monoclonal antibodiesincluding murine, fully humanized, and chimeric antibodies that havebeen approved by the FDA for therapeutic use. Rituximab and trastuzumabare among the top-selling protein therapeutics against cancer. Recently,monoclonal antibodies targeting immune checkpoint molecules, such asipilimumab (e.g., YERVOY) and nivolumab (e.g., OPDIVO®) have shownencouraging clinical results by inducing T cell immunity against tumors.As many patients do not respond well to monotherapy approaches,monoclonal antibodies are often combined with other immunomodulatoryapproaches, such as monoclonal antibodies against other targets, toenhance their efficacy. For example, clinical studies have demonstratedthat combination of nivolumab and ipilimumab results in improved ratesof objective response among melanoma patients.

With the development of molecular cloning technology and growingknowledge of antibody engineering, many formats have evolved to increasethe targeting capacity of therapeutic antibodies. Multispecific (such asbispecific) antibodies are designed to simultaneously modulate two ormore therapeutic targets in order to provide enhanced therapeuticefficacy and broadened potential utility. It has been reported thatbispecific antibodies can be more effective than simple combination oftwo monoclonal antibodies. A variety of multispecific antibody formatshave been developed. For example, bispecific antibodies have been madeby fusing antigen binding (Fab) fragments or single chain variablefragments (scFvs) to monoclonal antibodies (see, for example, Weidle etal. Cancer Genomics & Proteomics 2013; 10: 1-18). Bispecific T-cellengagers (BiTEs) have been developed using scFvs to bridge tumor cellswith immune cells and form an immunological synapse by taking advantageof their relatively small size. Bispecific antibodies in the IgG formatinclude asymmetric bispecific antibodies and homodimerized bispecificantibodies, all of which have an extended blood half-life and their owncrystalline fragment (Fc)-mediated functions. Multispecific antibodiesof different formats differ in size, are frequently produced bydifferent technologies, and have different in vivo distribution, tissuepenetration, and pharmacokinetic properties.

Despite their conceptual advantages, current bispecific antibodies arechallenging to manufacture and develop as biologic drugs. As artificialconstructs, bispecific antibodies cannot be produced by normal B-cells.Initial attempts to produce bispecific antibodies involved chemicalconjugation of monospecific antibodies and fusion of mAb-expressingcells, but these approaches suffer from low efficiency and the necessityof purification from abundant side products. Advanced methods in proteinengineering and molecular biology have enabled recombinant constructionof a variety of new bispecific antibody formats. However, once adoptedin these known engineered bispecific antibody formats, the individualcomponents, such as scFvs and mAbs, lose their favorable biochemicaland/or biophysical properties, serum half-life, and/or stability,resulting in poor efficacy, instability and high immunogenicity. See,for example, Fan G. et al. J. Hematol & Oncol, 2015; 8:130. Furthermore,many known bispecific antibody formats are associated with lowexpression levels that are impractical for industrial production. Thus,there remains a need for bispecific antibody platforms for practicalproduction and development into biologic drugs.

Single-domain antibodies (sdAbs) are antibody fragments each having asingle monomeric antibody variable domain. Despite their much smallersizes than common monoclonal antibodies having two heavy chains and twolight chains, sdAbs can bind antigens with similar affinity andspecificity as mAbs. Used as building blocks, the sdAbs can be fused toIgG Fc domains to create IgG-like antibodies, including bivalent andbispecific antibodies (see, for example, Hmila I. et al. Mol. Immunol.2008; 45: 3847-3856).

The disclosures of all publications, patents, patent applications andpublished patent applications referred to herein are hereby incorporatedherein by reference in their entirety.

BRIEF SUMMARY OF THE INVENTION

The present application provides a multispecific antigen binding protein(MABP) comprising one or more single-domain antibodies (sdAbs) fused toa full-length four-chain antibody or an antigen binding fragment derivedtherefrom.

Accordingly, one aspect of the present application provides a MABPcomprising: (a) a first antigen binding portion comprising a heavy chainvariable domain (V_(H)) and a light chain variable domain (V_(L)),wherein the V_(H) and V_(L) together form an antigen-binding site thatspecifically binds a first epitope, and (b) a second antigen bindingportion comprising a single-domain antibody (sdAb) that specificallybinds a second epitope, wherein the first antigen binding portion andthe second antigen binding portion are fused to each other. In someembodiments, the first epitope and the second epitope are from the sameantigen. In some embodiments, the first epitope and the second epitopeare from different antigens. In some embodiments, the MABP isbispecific.

In some embodiments according to any one of the MABPs described above,the first antigen binding portion is a full-length antibody consistingof two heavy chains and two light chains. In some embodiments, the firstantigen binding portion is an antibody fragment comprising a heavy chaincomprising the V_(H) and a light chain comprising the V_(L). In someembodiments, the second antigen binding portion comprises a singlepolypeptide chain. In some embodiments, the C terminus of the secondantigen binding portion is fused to the N-terminus of at least one heavychain of the first antigen binding portion. In some embodiments, the Cterminus of the second antigen binding portion is fused to theN-terminus of at least one light chain of the first antigen bindingportion. In some embodiments, the N terminus of the second antigenbinding portion is fused to the C-terminus of at least one heavy chainof the first antigen binding portion. In some embodiments, the Nterminus of the second antigen binding portion is fused to theC-terminus of at least one light chain of the first antigen bindingportion. In some embodiments, the second antigen binding portion is aFab-like domain comprising a first polypeptide chain comprising a firstsdAb fused to a C_(H)1 domain, and a second polypeptide chain comprisinga second sdAb fused to a C_(L) domain.

In some embodiments according to any one of the MABPs described above,the first antigen binding portion comprises a human, humanized orchimeric antibody or antigen binding fragment thereof.

In some embodiments according to any one of the MABPs described above,the first antigen binding portion comprises an Fc region. In someembodiments, the second antigen binding portion is fused to theN-terminus of the Fc region. In some embodiments, the Fc region is anIgG1 Fc. In some embodiments, the Fc region is an IgG4 Fc, such as anIgG4 Fc having an S228P mutation.

In some embodiments according to any one of the MABPs described above,the first antigen binding portion and the second antigen binding portionare fused to each other via a peptide bond or a peptide linker. In someembodiments, the peptide linker is no more than about 30 (such as nomore than about any one of 25, 20 or 15) amino acids long. In someembodiments, the peptide linker comprises the amino acid sequence of SEQID NO: 1, 8 or 13. In some embodiments, the first antigen bindingportion and the second antigen binding portion are fused to each otherchemically.

In some embodiments according to any one of the MABPs described above,the sdAb is a camelid, humanized, or human sdAb.

In some embodiments according to any one of the MABPs described above,the first epitope is from an immune checkpoint molecule. In someembodiments, the immune checkpoint molecule is selected from the groupconsisting of PD-1, PD-L1, PD-L2, CTLA-4, B7-H3, TIM-3, LAG-3, VISTA,ICOS, 4-1BB, OX40, GITR, and CD40. In some embodiments, the firstantigen binding portion is an anti-PD-1 antibody or antigen bindingfragment thereof. In some embodiments, the anti-PD-1 antibody isselected from the group consisting of pembrolizumab (e.g., KEYTRUDA) andnivolumab (e.g., OPVIDO®). In some embodiments, the first antigenbinding portion is an anti-PD-L1 antibody or antigen binding fragmentthereof. In some embodiments, the anti-PD-L1 antibody is duravalumab oratezolizumab. In some embodiments, the sdAb specifically binds an immunecheckpoint molecule, such as an immune checkpoint molecule selected fromthe group consisting of PD-1, PD-L1, PD-L2, CTLA-4, B7-H3, TIM-3, LAG-3,VISTA, ICOS, 4-1BB, OX40, GITR, and CD40. In some embodiments, thesecond antigen binding portion comprises an anti-CTLA-4 sdAb.

In some embodiments according to any one of the MABPs described above,the first epitope is from a tumor antigen. In some embodiments, thetumor antigen is selected from the group consisting of HER2, BRAF, EGFR,VEGFR2, CD20, RANKL, CD38, and CD52. In some embodiments, the firstantigen binding portion is an anti-HER2 antibody or antigen bindingfragment thereof. In some embodiments, the anti-HER2 antibody istrastuzumab. In some embodiments, the second antigen binding portioncomprises an anti-CD3 sdAb.

In some embodiments according to any one of the MABPs described above,the first epitope is from an angiogenic factor. In some embodiments, thefirst antigen binding portion is an anti-Ang2 antibody or antigenbinding fragment thereof, such as LC10. In some embodiments, the secondepitope is from a second angiogenic factor. In some embodiments, thesecond antigen binding portion is an anti-VEGF sdAb.

In some embodiments according to any one of the MABPs described above,the first epitope is from a pro-inflammatory molecule. In someembodiments, the pro-inflammatory molecule is selected from the groupconsisting of IL-1β, TNF-α, IL-5, IL-6, IL-6R, and eotaxin-1. In someembodiments, the first antigen binding portion is an anti-TNF-α antibodyor antigen binding fragment thereof. In some embodiments, the anti-TNF-αantibody is adalimumab. In some embodiments, the second antigen bindingportion comprises an anti-IL-1β sdAb. In some embodiments, the firstantigen binding portion is an anti-IL-5 antibody or antigen bindingfragment thereof. In some embodiments, the anti-IL-5 antibody ismepolizumab. In some embodiments, the second antigen binding portioncomprises an anti-eotaxin-1 sdAb.

In some embodiments according to any one of the MABPs described above,the MABP can be produced recombinantly, such as in mammalian cells(e.g., CHO cells), at an expression level of at least about 10 mg/L,such as at least about 10 mg/L, 15 mg/L, 50 mg/mL, or higher. In someembodiments, the MABP has a solubility of at least about 100 mg/mL, suchas at least about 150 mg/mL, 200 mg/mL or higher. In some embodiments,the MABP has an aggregation onset temperature (T_(agg)) of at leastabout 65° C., such as about 65° C. to about 75° C. In some embodiments,the MABP has an unfolding midpoint temperature (T_(m)) of at least about65° C., such as about 65° C. to about 75° C. In some embodiments, theMABP is stable for at least about one week at 25° C. at a concentrationof at least about 50 mg/mL. In some embodiments, the MABP is stable forat least about one week at 37° C. at a concentration of at least about50 mg/mL. In some embodiments, the MABP is stable after at least about 5freeze-thaw cycles at a concentration of at least 50 mg/mL.

Another aspect of the present application provides a pharmaceuticalcomposition comprising any one of the MABPs described above and apharmaceutically acceptable carrier. In some embodiments, theconcentration of the MABP is at least about 100 mg/mL, such as at leastabout 150 mg/mL, 200 mg/mL or higher.

Further provided in one aspect of the present application is a method oftreating a disease in an individual, comprising administering to theindividual an effective amount of any one of the pharmaceuticalcompositions described above. In some embodiments, the disease is acancer. In some embodiments, the cancer is selected from the groupconsisting of breast cancer, renal cancer, melanoma, lung cancer,glioblastoma, head and neck cancer, prostate cancer, ovarian carcinoma,bladder carcinoma, and lymphoma. In some embodiments, the disease is aninflammatory or autoimmune disease. In some embodiments, theinflammatory or autoimmune disease is selected from the group consistingof arthritis (such as rheumatoid arthritis, juvenile idiopathicarthritis, psoriatic arthritis, ankylosing spondylitis, and arthriticulcerative colitis), colitis, psoriasis, severe asthma, and moderate tosevere Crohn's disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a schematic structure of an exemplary bispecific antigenbinding protein (also referred herein as “BABP”) comprising amonospecific full-length antibody having two identical heavy chains andtwo identical light chains, and an sdAb, wherein the N-terminus of thesdAb is fused to the C-terminus of one heavy chain via an optionalpeptide linker. The full-length antibody has two antigen binding sitesthat specifically bind the first epitope. The sdAb specifically bindsthe second epitope. For example, the BABP can consist of fourpolypeptide chains with structures from the N-terminus to the C-terminusas follows: (1) V_(L)-C_(L); (2) V_(H)-C_(H)1-C_(H)2-C_(H)3-V_(H)H; (3)V_(H)-C_(H)1-C_(H)2-C_(H)3; and (4) V_(L)-C_(L), wherein V_(H) and V_(L)of polypeptide chains (1) and (2) forms an antigen binding site thatspecifically binds a first copy of the first epitope, V_(H) and V_(L) ofpolypeptide chains (3) and (4) forms an antigen binding site thatspecifically binds a second copy of the first epitope, and V_(H)Hspecifically binds the second epitope.

FIG. 2 depicts a schematic structure of an exemplary BABP comprising amonospecific full-length antibody having two identical heavy chains andtwo identical light chains, and two identical sdAbs, wherein the twosdAbs are fused to each other, and the N-terminus of one sdAb is fusedto the C-terminus of a heavy chain via an optional peptide linker. Thefull-length antibody has two antigen binding sites that specificallybind the first epitope. The two sdAbs specifically bind the secondepitope. For example, the BABP can consist of four polypeptide chainswith structures from the N-terminus to the C-terminus as follows: (1)V_(L)-C_(L); (2) V_(H)-C_(H)1-C_(H)2-C_(H)3-V_(H)H-V_(H)H; (3)V_(H)-C_(H)1-C_(H)2-C_(H)3; and (4) V_(L)-C_(L), wherein V_(H) and V_(L)of polypeptide chains (1) and (2) forms an antigen binding site thatspecifically binds a first copy of the first epitope, V_(H) and V_(L) ofpolypeptide chains (3) and (4) forms an antigen binding site thatspecifically binds a second copy of the first epitope, and each V_(H)Hspecifically binds a copy of the second epitope.

FIG. 3 depicts a schematic structure of an exemplary trispecific antigenbinding protein (also referred herein as “TABP”) comprising amonospecific full-length antibody having two identical heavy chains andtwo identical light chains, a first sdAb, and a second sdAb, wherein thefirst sdAb and the second sdAb are fused to each other via an optionalpeptide linker, and the N-terminus of the first sdAb is fused to theC-terminus of a heavy chain via an optional peptide linker. Thefull-length antibody has two antigen binding sites that specificallybind the first epitope. The first sdAb specifically binds the secondepitope. The second sdAb specifically binds the third epitope. Forexample, the TABP can consist of four polypeptide chains with structuresfrom the N-terminus to the C-terminus as follows: (1) V_(L)-C_(L); (2)V_(H)-C_(H)1-C_(H)2-C_(H)3-V_(H)H1-V_(H)H2; (3)V_(H)-C_(H)1-C_(H)2-C_(H)3; and (4) V_(L)-C_(L), wherein V_(H) and V_(L)of polypeptide chains (1) and (2) forms an antigen binding site thatspecifically binds a first copy of the first epitope, V_(H) and V_(L) ofpolypeptide chains (3) and (4) forms an antigen binding site thatspecifically binds a second copy of the first epitope, V_(H)H1specifically binds the second epitope, and V_(H)H2 specifically bindsthe third epitope.

FIG. 4 depicts a schematic structure of an exemplary BABP comprising amonospecific full-length antibody having two identical heavy chains andtwo identical light chains, and two identical sdAbs, wherein theN-terminus of each sdAb is fused to the C terminus of one heavy chainvia an optional peptide linker. The full-length antibody has two antigenbinding sites that specifically bind a first epitope. The two sdAbsspecifically bind the second epitope. For example, the BABP can consistof four polypeptide chains with structures from the N-terminus to theC-terminus as follows: (1) V_(L)-C_(L); (2)V_(H)-C_(H)1-C_(H)2-C_(H)3-V_(H)H; (3)V_(H)-C_(H)1-C_(H)2-C_(H)3-V_(H)H; and (4) V_(L)-C_(L), wherein V_(H)and V_(L) of polypeptide chains (1) and (2) forms an antigen bindingsite that specifically binds a first copy of the first epitope, V_(H)and V_(L) of polypeptide chains (3) and (4) forms an antigen bindingsite that specifically binds a second copy of the first epitope, andeach V_(H)H specifically binds a copy of the second epitope. Inalternative formats, each sdAb may be replaced with two copies of thesdAb fused to each other.

FIG. 5 depicts a schematic structure of an exemplary BABP comprising amonospecific Fab having a heavy chain and a light chain, and twoidentical sdAbs, wherein the N-terminus of an sdAb is fused to theC-terminus of the heavy chain via an optional peptide linker, and theother sdAb is fused to the C-terminus of the light chain of the Fab viaan optional peptide linker. The Fab specifically binds the firstepitope. The two sdAbs specifically bind the second epitope. Forexample, the BABP can consist of two polypeptide chains with structuresfrom the N-terminus to the C-terminus as follows: (1)V_(L)-C_(L)-V_(H)H; and (2) V_(H)-C_(H)1-V_(H)H, wherein V_(H) and V_(L)of polypeptide chains (1) and (2) forms an antigen binding site thatspecifically binds the first epitope, and each V_(H)H specifically bindsa copy of the second epitope. In alternative formats, each sdAb may beomitted, or replaced with two identical or different sdAbs fused to eachother.

FIG. 6 depicts a schematic structure of an exemplary TABP comprising abispecific full-length antibody having two heavy chains and two lightchains, and two identical sdAbs, wherein the N-terminus of each sdAb isfused to one heavy chain via an optional peptide linker. The full-lengthantibody has a first antigen binding site that specifically binds thefirst epitope, and a second antigen binding site that specifically bindsthe third epitope. The two sdAbs specifically bind to the secondepitope. For example, the TABP can consist of four polypeptide chainswith structures from the N-terminus to the C-terminus as follows: (1)V_(L)1-C_(L); (2) V_(H)1-C_(H)1-C_(H)2-C_(H)3-V_(H)H; (3)V_(H)2-C_(H)1-C_(H)2-C_(H)3-V_(H)H; and (4) V_(L)2-C_(L), wherein V_(H)1and V_(L)1 of polypeptide chains (1) and (2) forms an antigen bindingsite that specifically binds the first epitope, V_(H)2 and V_(L)2 ofpolypeptide chains (3) and (4) forms an antigen binding site thatspecifically binds the third epitope, and each V_(H)H specifically bindsa copy of the second epitope. In alternative formats, each sdAb may beomitted, or replaced with two identical or different sdAbs fused to eachother.

FIG. 7 depicts a schematic structure of an exemplary TABP comprising amonospecific full-length antibody having two identical heavy chains andtwo identical light chains, a first sdAb, and a second sdAb, wherein theN-terminus of each sdAb is fused to one heavy chain via an optionalpeptide linker. The full-length antibody has two antigen binding sitesthat specifically bind the first epitope. The first sdAb specificallybinds the second epitope. The second sdAb specifically binds the thirdepitope. For example, the TABP can consist of four polypeptide chainswith structures from the N-terminus to the C-terminus as follows: (1)V_(L)-C_(L); (2) V_(H)-C_(H)1-C_(H)2-C_(H)3-V_(H)H1; (3)V_(H)-C_(H)1-C_(H)2-C_(H)3-V_(H)H2; and (4) V_(L)-C_(L), wherein V_(H)and V_(L) of polypeptide chains (1) and (2) forms an antigen bindingsite that specifically binds a first copy of the first epitope, V_(H)and V_(L) of polypeptide chains (3) and (4) forms an antigen bindingsite that specifically binds a second copy of the first epitope, V_(H)H1specifically binds the second epitope, and V_(H)H2 specifically bindsthe third epitope. In alternative formats, each sdAb may be omitted, orreplaced with two identical or different sdAbs fused to each other. Themonospecific full-length antibody may be replaced with a bispecificfull-length antibody to further expand binding specificity.

FIG. 8 depicts a schematic structure of an exemplary tetraspecificantigen binding protein comprising a bispecific full-length antibodyhaving two heavy chains and two light chains, a first sdAb, and a secondsdAb, wherein the N-terminus of each sdAb is fused to one heavy chainvia an optional peptide linker. The full-length antibody has a firstantigen binding site that specifically binds the first epitope, and asecond antigen binding site that specifically binds the third epitope.The first sdAb specifically binds the second epitope. The second sdAbspecifically binds the fourth epitope. For example, the tetraspecificantigen binding protein can consist of four polypeptide chains withstructures from the N-terminus to the C-terminus as follows: (1)V_(L)1-C_(L); (2) V_(H)1-C_(H)1-C_(H)2-C_(H)3-V_(H)H1; (3)V_(H)2-C_(H)1-C_(H)2-C_(H)3-V_(H)H2; and (4) V_(L)2-C_(L), whereinV_(H)1 and V_(L)1 of polypeptide chains (1) and (2) forms an antigenbinding site that specifically binds the first epitope, V_(H)2 andV_(L)2 of polypeptide chains (3) and (4) forms an antigen binding sitethat specifically binds the third epitope, V_(H)H1 specifically bindsthe second epitope, and V_(H)H2 specifically binds the fourth epitope.In alternative formats, each sdAb may be omitted, or replaced with twoidentical or different sdAbs fused to each other.

FIG. 9 depicts a schematic structure of an exemplary BABP comprising amonospecific full-length antibody having two identical heavy chains andtwo identical light chains, and two identical sdAbs, wherein theC-terminus of each sdAb is fused to the N-terminus of one heavy chain.The full-length antibody has two antigen binding sites that specificallybind a first epitope. The two sdAbs specifically bind the secondepitope. For example, the BABP can consist of four polypeptide chainswith structures from the N-terminus to the C-terminus as follows: (1)V_(L)-C_(L); (2) V_(H)H-V_(H)-C_(H)1-C_(H)2-C_(H)3; (3)V_(H)H-V_(H)-C_(H)1-C_(H)2-C_(H)3; and (4) V_(L)-C_(L), wherein V_(H)and V_(L) of polypeptide chains (1) and (2) forms an antigen bindingsite that specifically binds a first copy of the first epitope, V_(H)and V_(L) of polypeptide chains (3) and (4) forms an antigen bindingsite that specifically binds a second copy of the first epitope, andeach V_(H)H specifically binds a copy of the second epitope. Inalternative formats, each sdAb may be omitted, or replaced with twoidentical or different sdAbs fused to each other. The monospecificfull-length antibody may be replaced with a bispecific full-lengthantibody to further expand binding specificity.

FIG. 10 depicts a schematic structure of an exemplary TABP comprising amonospecific full-length antibody having two identical heavy chains andtwo identical light chains, a first sdAb, and a second sdAb, wherein theC-terminus of each sdAb is fused to the N-terminus of one heavy chain.The full-length antibody has two antigen binding sites that specificallybind the first epitope. The first sdAb specifically binds the secondepitope. The second sdAb specifically binds the third epitope. Forexample, the TABP can consist of four polypeptide chains with structuresfrom the N-terminus to the C-terminus as follows: (1) V_(L)-C_(L); (2)V_(H)H1-V_(H)-C_(H)1-C_(H)2-C_(H)3; (3)V_(H)H2-V_(H)-C_(H)1-C_(H)2-C_(H)3; and (4) V_(L)-C_(L), wherein V_(H)and V_(L) of polypeptide chains (1) and (2) forms an antigen bindingsite that specifically binds a first copy of the first epitope, V_(H)and V_(L) of polypeptide chains (3) and (4) forms an antigen bindingsite that specifically binds a second copy of the first epitope, V_(H)H1specifically binds the second epitope, and V_(H)H2 specifically bindsthe third epitope. In alternative formats, each sdAb may be omitted, orreplaced with two identical or different sdAbs fused to each other. Themonospecific full-length antibody may be replaced with a bispecificfull-length antibody to further expand binding specificity.

FIG. 11 depicts a schematic structure of an exemplary BABP comprising amonospecific full-length antibody having two identical heavy chains andtwo identical light chains, and two identical sdAbs, wherein theN-terminus of each sdAb is fused to the C-terminus of one light chainvia an optional peptide linker. The full-length antibody has two antigenbinding sites that specifically bind a first epitope. The two sdAbsspecifically bind the second epitope. For example, the BABP can consistof four polypeptide chains with structures from the N-terminus to theC-terminus as follows: (1) V_(L)-C_(L)-V_(H)H; (2)V_(H)-C_(H)1-C_(H)2-C_(H)3; (3) V_(H)-C_(H)1-C_(H)2-C_(H)3; and (4)V_(L)-C_(L)-V_(H)H, wherein V_(H) and V_(L) of polypeptide chains (1)and (2) forms an antigen binding site that specifically binds a firstcopy of the first epitope, V_(H) and V_(L) of polypeptide chains (3) and(4) forms an antigen binding site that specifically binds a second copyof the first epitope, and each V_(H)H specifically binds a copy of thesecond epitope. In alternative formats, each sdAb may be omitted, orreplaced with two identical or different sdAbs fused to each other. Themonospecific full-length antibody may be replaced with a bispecificfull-length antibody to further expand binding specificity.

FIG. 12 depicts a schematic structure of an exemplary TABP comprising amonospecific full-length antibody having two identical heavy chains andtwo identical light chains, a first sdAb, and a second sdAb, wherein theN-terminus of each sdAb is fused to the C-terminus of one light chainvia an optional peptide linker. The full-length antibody has two antigenbinding sites that specifically bind a first epitope. The first sdAbspecifically binds the second epitope. The second sdAb specificallybinds the third epitope. For example, the TABP can consist of fourpolypeptide chains with structures from the N-terminus to the C-terminusas follows: (1) V_(L)-C_(L)-V_(H)H1; (2) V_(H)-C_(H)1-C_(H)2-C_(H)3; (3)V_(H)-C_(H)1-C_(H)2-C_(H)3; and (4) V_(L)-C_(L)-V_(H)H2, wherein V_(H)and V_(L) of polypeptide chains (1) and (2) forms an antigen bindingsite that specifically binds a first copy of the first epitope, V_(H)and V_(L) of polypeptide chains (3) and (4) forms an antigen bindingsite that specifically binds a second copy of the first epitope, V_(H)H1specifically binds the second epitope, and V_(H)H2 specifically bindsthe third epitope. In alternative formats, each sdAb may be omitted, orreplaced with two identical or different sdAbs fused to each other. Themonospecific full-length antibody may be replaced with a bispecificfull-length antibody to further expand binding specificity.

FIG. 13 depicts a schematic structure of an exemplary BABP comprising amonospecific full-length antibody having two identical heavy chains andtwo identical light chains, and two identical sdAbs, wherein theC-terminus of each sdAb is fused to the N-terminus of one light chainvia an optional peptide linker. The full-length antibody has two antigenbinding sites that specifically bind a first epitope. The two sdAbsspecifically bind the second epitope. For example, the BABP can consistof four polypeptide chains with structures from the N-terminus to theC-terminus as follows: (1) V_(H)H-V_(L)-C_(L); (2)V_(H)-C_(H)1-C_(H)2-C_(H)3; (3) V_(H)-C_(H)1-C_(H)2-C_(H)3; and (4)V_(H)H-V_(L)-C_(L), wherein V_(H) and V_(L) of polypeptide chains (1)and (2) forms an antigen binding site that specifically binds a firstcopy of the first epitope, V_(H) and V_(L) of polypeptide chains (3) and(4) forms an antigen binding site that specifically binds a second copyof the first epitope, and each V_(H)H specifically binds a copy of thesecond epitope. In alternative formats, each sdAb may be omitted, orreplaced with two identical or different sdAbs fused to each other. Themonospecific full-length antibody may be replaced with a bispecificfull-length antibody to further expand binding specificity.

FIG. 14 depicts a schematic structure of an exemplary TABP comprising amonospecific full-length antibody having two identical heavy chains andtwo identical light chains, a first sdAb, and a second sdAb, wherein theC-terminus of each sdAb is fused to the N-terminus of one light chainvia an optional peptide linker. The full-length antibody has two antigenbinding sites that specifically bind a first epitope. The first sdAbspecifically binds the second epitope. The second sdAb specificallybinds the third epitope. For example, the TABP can consist of fourpolypeptide chains with structures from the N-terminus to the C-terminusas follows: (1) V_(H)H1-V_(L)-C_(L); (2) V_(H)-C_(H)1-C_(H)2-C_(H)3; (3)V_(H)-C_(H)1-C_(H)2-C_(H)3; and (4) V_(H)H2-V_(L)- C_(L), wherein V_(H)and V_(L) of polypeptide chains (1) and (2) forms an antigen bindingsite that specifically binds a first copy of the first epitope, V_(H)and V_(L) of polypeptide chains (3) and (4) forms an antigen bindingsite that specifically binds a second copy of the first epitope, V_(H)H1specifically binds the second epitope, and V_(H)H2 specifically bindsthe third epitope. In alternative formats, each sdAb may be omitted, orreplaced with two identical or different sdAbs fused to each other. Themonospecific full-length antibody may be replaced with a bispecificfull-length antibody to further expand binding specificity.

FIG. 15 depicts a schematic structure of an exemplary TABP comprising amonospecific full-length antibody having two identical heavy chains andtwo identical light chains, two identical first sdAbs, and two identicalsecond sdAbs, wherein the C-terminus of each first sdAb is fused to theN-terminus of one heavy chain via an optional peptide linker, and theN-terminus of each second sdAb is fused to the C-terminus of one heavychain via an optional peptide linker. The full-length antibody has twoantigen binding sites that specifically bind a first epitope. The firstsdAb specifically binds the second epitope. The second sdAb specificallybinds the third epitope. For example, the TABP can consist of fourpolypeptide chains with structures from the N-terminus to the C-terminusas follows: (1) V_(L)-C_(L); (2)V_(H)H1-V_(H)-C_(H)1-C_(H)2-C_(H)3-V_(H)H2; (3)V_(H)H1-V_(H)-C_(H)1-C_(H)2-C_(H)3-V_(H)H2; and (4) V_(L)-C_(L), whereinV_(H) and V_(L) of polypeptide chains (1) and (2) forms an antigenbinding site that specifically binds a first copy of the first epitope,V_(H) and V_(L) of polypeptide chains (3) and (4) forms an antigenbinding site that specifically binds a second copy of the first epitope,each V_(H)H1 specifically binds a copy of the second epitope, and eachV_(H)H2 specifically binds a copy of the third epitope. In alternativeformats, each sdAb may be omitted, or replaced with two identical ordifferent sdAbs fused to each other. The monospecific full-lengthantibody may be replaced with a bispecific full-length antibody tofurther expand binding specificity.

FIG. 16 depicts a schematic structure of an exemplary TABP comprising amonospecific full-length antibody having two identical heavy chains andtwo identical light chains, two identical first sdAbs, and two identicalsecond sdAbs, wherein the C-terminus of each first sdAb is fused to theN-terminus of one light chain via an optional peptide linker, and theN-terminus of each second sdAb is fused to the C-terminus of one heavychain via an optional peptide linker. The full-length antibody has twoantigen binding sites that each specifically binds a first epitope. Thefirst sdAb specifically binds a second epitope. The second sdAbspecifically binds a third epitope. For example, the TABP can consist offour polypeptide chains with structures from the N-terminus to theC-terminus as follows: (1) V_(H)H1-V_(L)-C_(L); (2)V_(H)-C_(H)1-C_(H)2-C_(H)3-V_(H)H2; (3)V_(H)-C_(H)1-C_(H)2-C_(H)3-V_(H)H2; and (4) V_(H)H1-V_(L)-C_(L), whereinV_(H) and V_(L) of polypeptide chains (1) and (2) forms an antigenbinding site that specifically binds a first copy of the first epitope,V_(H) and V_(L) of polypeptide chains (3) and (4) forms an antigenbinding site that specifically binds a second copy of the first epitope,each V_(H)H1 specifically binds a copy of the second epitope, and eachV_(H)H2 specifically binds a copy of the third epitope. In alternativeformats, each sdAb may be omitted, or replaced with two identical ordifferent sdAbs fused to each other. The monospecific full-lengthantibody may be replaced with a bispecific full-length antibody tofurther expand binding specificity.

FIG. 17 depicts a schematic structure of an exemplary BABP comprising amonospecific full-length antibody having two identical heavy chains andtwo identical light chains, and four identical sdAbs, wherein theC-terminus of each sdAb is fused to the N-terminus of heavy chain orlight chain of the monospecific full-length antibody via an optionalpeptide linker. The full-length antibody has two antigen binding sitesthat each specifically binds a first epitope. Each sdAb specificallybinds to a second epitope. For example, the BABP can consist of fourpolypeptide chains with structures from the N-terminus to the C-terminusas follows: (1) V_(H)H-V_(L)-C_(L); (2)V_(H)H-V_(H)-C_(H)1-C_(H)2-C_(H)3; (3)V_(H)H-V_(H)-C_(H)1-C_(H)2-C_(H)3; and (4) V_(H)H-V_(L)-C_(L), whereinV_(H) and V_(L) of polypeptide chains (1) and (2) forms an antigenbinding site that specifically binds a first copy of the first epitope,V_(H) and V_(L) of polypeptide chains (3) and (4) forms an antigenbinding site that specifically binds a second copy of the first epitope,and each V_(H)H specifically binds a copy of the second epitope. Inalternative formats, each sdAb may be omitted, or replaced with twoidentical or different sdAbs fused to each other. The monospecificfull-length antibody may be replaced with a bispecific full-lengthantibody to further expand binding specificity.

FIG. 18 depicts a schematic structure of an exemplary BABP comprising amonospecific full-length antibody having two identical heavy chains andtwo identical light chains, and four identical sdAbs, wherein fused tothe N-terminus of each heavy chain are two identical sdAbs, and the twosdAbs are fused to each other via an optional peptide linker. Thefull-length antibody has two antigen binding sites that eachspecifically binds a first epitope. Each sdAb specifically binds asecond epitope. For example, the BABP can consist of four polypeptidechains with structures from the N-terminus to the C-terminus as follows:(1) V_(L)-C_(L); (2) V_(H)H-V_(H)H-V_(H)-C_(H)1-C_(H)2-C_(H)3; (3)V_(H)H-V_(H)H-V_(H)-C_(H)1-C_(H)2-C_(H)3; and (4) V_(L)-C_(L), whereinV_(H) and V_(L) of polypeptide chains (1) and (2) forms an antigenbinding site that specifically binds a first copy of the first epitope,V_(H) and V_(L) of polypeptide chains (3) and (4) forms an antigenbinding site that specifically binds a second copy of the first epitope,and each V_(H)H specifically binds a copy of the second epitope. Inalternative formats, each sdAb may be omitted, or replaced with twoidentical or different sdAbs fused to each other. The monospecificfull-length antibody may be replaced with a bispecific full-lengthantibody to further expand binding specificity.

FIG. 19 depicts a schematic structure of an exemplary BABP comprising amonospecific full-length antibody having two identical heavy chains andtwo identical light chains, and two identical sdAbs, wherein theN-terminus of each sdAb is fused to the C-terminus of the CH1 region viaan optional peptide linker and C-terminus of each sdAb is fused to theN-terminus of the CH2 region of the monospecific full-length antibody.The full-length antibody has two antigen binding sites that eachspecifically binds a first epitope. Each sdAb specifically binds asecond epitope. For example, the BABP can consist of four polypeptidechains with structures from the N-terminus to the C-terminus as follows:(1) V_(L)-C_(L); (2) V_(H)-C_(H)1-V_(H)H-C_(H)2-C_(H)3; (3)V_(H)-C_(H)1-V_(H)H-C_(H)2-C_(H)3; and (4) V_(L)-C_(L), wherein V_(H)and V_(L) of polypeptide chains (1) and (2) forms an antigen bindingsite that specifically binds a first copy of the first epitope, V_(H)and V_(L) of polypeptide chains (3) and (4) forms an antigen bindingsite that specifically binds a second copy of the first epitope, andeach V_(H)H specifically binds a copy of the second epitope. Inalternative formats, each sdAb may be omitted, or replaced with twoidentical or different sdAbs fused to each other. The monospecificfull-length antibody may be replaced with a bispecific full-lengthantibody to further expand binding specificity. In alternative formats,to expand specificity, the two Fab fragments can specifically binddifferent epitopes, and/or the V_(H)H fragments can specifically binddifferent epitopes.

FIG. 20 depicts a schematic structure of an exemplary BABP comprisingtwo identical single chain variable fragments (scFvs), two identicalsdAbs and a fragment crystallizable (Fc) region, wherein the N-terminusof each sdAb is fused to the C-terminus of an scFv via an optionalpeptide linker and the C-terminus of each sdAb is fused to theN-terminus of the Fc region. Each scFv specifically binds a firstepitope. Each sdAb specifically binds a second epitope. For example, theBABP can consist of two polypeptide chains each with a structure fromthe N-terminus to the C-terminus as follows:V_(L)-V_(H)-V_(H)H-C_(H)2-C_(H)3, wherein V_(H) and V_(L) of eachpolypeptide chain forms a scFv domain that specifically binds a copy ofthe first epitope, and each V_(H)H specifically binds a copy of thesecond epitope. In alternative formats, the scFv domain can comprisefrom the N-terminus to the C-termins: V_(H)-V_(L). Additionally, toexpand specificity, the two scFvs can specifically bind differentepitopes, and/or the V_(H)H fragments can specifically bind differentepitopes.

FIG. 21 depicts a schematic structure of an exemplary BABP comprisingtwo identical antigen-binding (Fab) fragments, two identical Fab-likefragments each comprising two V_(H)H fragments, and an Fc region. Ineach Fab-like domain, the V_(H) and V_(L) regions are each replaced byan sdAb. Each Fab fragment specifically binds a first epitope, and eachFab-like fragment specifically binds a second epitope. For example, theBABP can consist of four polypeptide chains with structures from theN-terminus to the C-terminus as follows: (1) V_(L)-C_(L)-V_(H)H-C_(L);(2) V_(H)-C_(H)1-V_(H)H-C_(H)1-C_(H)2-C_(H)3; (3)V_(H)-C_(H)1-V_(H)H-C_(H)1-C_(H)2-C_(H)3; and (4)V_(L)-C_(L)-V_(H)H-C_(L), wherein V_(H) and V_(L) of polypeptide chains(1) and (2) forms an antigen binding site that specifically binds afirst copy of the first epitope, V_(H) and V_(L) of polypeptide chains(3) and (4) forms an antigen binding site that specifically binds asecond copy of the first epitope, and each V_(H)H specifically binds acopy of the second epitope. In alternative formats, to expandspecificity, the two Fab fragments can specifically bind differentepitopes, and/or the Fab-like fragments can specifically bind differentepitopes.

FIG. 22 depicts a schematic structure of an exemplary BABP comprisingtwo identical scFvs, two identical Fab-like fragments each comprisingtwo V_(H)H fragments, and an Fc region. In each Fab-like domain, theV_(H) and V_(L) regions are each replaced by an sdAb. For example, theBABP can consist of four polypeptide chains with structures from theN-terminus to the C-terminus as follows: (1) V_(H)H-C_(L); (2)V_(L)-V_(H)-V_(H)H-C_(H)1-C_(H)2-C_(H)3; (3)V_(L)-V_(H)-V_(H)H-C_(H)1-C_(H)2-C_(H)3; and (4) V_(H)H-C_(L), whereinV_(H) and V_(L) of polypeptide chains (2) and (3) each forms an scFvthat specifically binds a copy of the first epitope, and each V_(H)Hspecifically binds a copy of the second epitope. In alternative formats,the C-terminus of the scFv may be fused to the N-terminus of the chainin the Fab-like fragment comprising V_(H)H-C_(L); and/or the scFv domaincan comprise from the N-terminus to the C-termins: V_(H)-V_(L).Additionally, to expand specificity, the two scFvs can specifically binddifferent epitopes, and/or the V_(H)H fragments can specifically binddifferent epitopes.

FIG. 23 shows the results from an in vivo efficacy experiment of BABPsBCP-75 and BCP-79 in MC38 syngeneic model in C56BL/6 PD-1 KI mice. Theresults of the BABPs are compared to those of the two backbone 4-chainantibodies, in-house expressed biosimilar antibodies pembrolizumab andnivolumab.

FIG. 24 shows the results from an in vivo efficacy experiment of BABPsBCP-75 and BCP-79 in MC38 syngeneic model in C56BL/6 CTLA-4 KI mice. Theresults of the BABPs are compared to those of Fc fusion proteinscomprising sdAb-2 or sdAb-3, wherein the Fc fragment is the same as thein-house expressed biosimilar antibodies pembrolizumab and nivolumab.In-house expressed ipilimumab of the IgG1 isotype serves as the positivecontrol for this experiment.

FIG. 25 shows the results from an in vivo efficacy experiment of BABPsBCP-84 and BCP-85 compared to combination therapy in human PD-L1 KI MC38syngeneic model in C56BL/6 CTLA-4 KI mice.

FIG. 26A and FIG. 26B show the results from an in vivo efficacyexperiment of BABP BCP-49 compared to combination therapy in A431xenograft model in BALB/c nude mice.

DETAILED DESCRIPTION OF THE INVENTION

The present application provides a MABP comprising a single-domainantibody (sdAb) fused to a full-length antibody or antigen bindingfragment that comprise a heavy chain variable domain (V_(H)) and a lightchain variable domain (V_(L)). The sdAb specifically binds a target(such as an epitope or antigen) that is distinct from the target(s)recognized by the full-length antibody or antigen binding fragment,thereby conferring a broadened targeting capability. As a building blockin a MABP, sdAb has several advantages over other antigen bindingfragments such as Fab and scFv used in currently known multispecificantibody formats, including, but not limited to, small size, highsolubility and stability, weak immunogenicity in human, and ability totarget a variety of epitopes. Thus, the MABPs described herein can havesimilar molecular weight and pharmacokinetic properties compared tothose of the full-length antibody or antigen binding fragment component.For example, a MABP can be designed by fusing one or more sdAbs to amonoclonal antibody with proven clinical efficacy and safety to provideincreased clinical benefits and desirable pharmacokinetic propertieswithout impeding the expressibility of the multispecific construct. Insome embodiments, the MABP comprises two naturally produced componentsor derivatives thereof, e.g. a naturally produced or humanized CamelidV_(H)H fragment, and a naturally produced monoclonal antibody, fused toeach other by polypeptide linkers. Unlike the majority of knownbispecific antibody formats, the MABP of the present application hasexcellent productivity, stability and solubility. In vitro efficacy datafurther indicates that the MABP retains anti-tumor activity of theparental antibodies. Synergistic activity are also found or expected inin vivo tumor animal models. The MABP format of the present applicationcan be adopted to target a variety of disease-related epitope or antigencombinations, such as a combination of immune checkpoint molecules, acombination of cell surface antigens (such as tumor antigens), or acombination of pro-inflammatory molecules, thereby providing agents thatare useful for treating a variety of diseases and conditions, such ascancer, inflammation, and autoimmune diseases.

Accordingly, one aspect of the present application provides a MABPcomprising: (a) a first antigen binding portion comprising a heavy chainvariable domain (V_(H)) and a light chain variable domain (V_(L)),wherein the V_(H) and V_(L) together form an antigen-binding site thatspecifically binds a first epitope, and (b) a second antigen bindingportion comprising an sdAb that specifically binds a second epitope,wherein the first antigen binding portion and the second antigen bindingportion are fused to each other.

One aspect of the present application provides a MABP comprising: (a) afirst antigen binding portion comprising a heavy chain variable domain(V_(H)) and a light chain variable domain (V_(L)), wherein the V_(H) andV_(L) together form an antigen-binding site that specifically binds afirst immune checkpoint molecule, and (b) a second antigen bindingportion comprising an sdAb that specifically binds a second immunecheckpoint molecule, wherein the first antigen binding portion and thesecond antigen binding portion are fused to each other.

One aspect of the present application provides a MABP comprising: (a) afirst antigen binding portion comprising a heavy chain variable domain(V_(H)) and a light chain variable domain (V_(L)), wherein the V_(H) andV_(L) together form an antigen-binding site that specifically binds afirst pro-inflammatory molecule, and (b) a second antigen bindingportion comprising an sdAb that specifically binds a secondpro-inflammatory molecule, wherein the first antigen binding portion andthe second antigen binding portion are fused to each other.

One aspect of the present application provides a MABP comprising: (a) afirst antigen binding portion comprising a heavy chain variable domain(V_(H)) and a light chain variable domain (V_(L)), wherein the V_(H) andV_(L) together form an antigen-binding site that specifically binds afirst tumor antigen, and (b) a second antigen binding portion comprisingan sdAb that specifically binds a cell surface antigen (such as tumorantigen, or a cell surface antigen on an immune effector cell), whereinthe first antigen binding portion and the second antigen binding portionare fused to each other.

Also provided are pharmaceutical compositions, kits and articlesmanufacture comprising the MABPs, and methods of treating a diseaseusing the MABPs described herein.

I. Definitions

The practice of the present invention will employ, unless indicatedspecifically to the contrary, conventional methods of virology,immunology, microbiology, molecular biology and recombinant DNAtechniques within the skill of the art, many of which are describedbelow for the purpose of illustration. Such techniques are explainedfully in the literature. See, e.g., Current Protocols in MolecularBiology or Current Protocols in Immunology, John Wiley & Sons, New York,N.Y. (2009); Ausubel et al, Short Protocols in Molecular Biology, 3^(rd)ed., Wiley & Sons, 1995; Sambrook and Russell, Molecular Cloning: ALaboratory Manual (3rd Edition, 2001); Maniatis et al. MolecularCloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach,vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed.,1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985);Transcription and Translation (B. Hames & S. Higgins, eds., 1984);Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guideto Molecular Cloning (1984) and other like references.

As used herein, the term “treatment” refers to clinical interventiondesigned to alter the natural course of the individual or cell beingtreated during the course of clinical pathology. Desirable effects oftreatment include decreasing the rate of disease progression,ameliorating or palliating the disease state, and remission or improvedprognosis. For example, an individual is successfully “treated” by theMABP of the present application if one or more symptoms associated withthe disease or condition being treated (such as cancer, inflammatory orautoimmune disease) are mitigated or eliminated.

As used herein, an “effective amount” refers to an amount of an agent ordrug effective to treat a disease or condition in a subject. In the caseof cancer, the effective amount of the MABP of the present applicationmay reduce the number of cancer cells; reduce the tumor size; inhibit(i.e., slow to some extent and preferably stop) cancer cell infiltrationinto peripheral organs; inhibit (i.e., slow to some extent andpreferably stop) tumor metastasis; inhibit, to some extent, tumorgrowth; and/or relieve to some extent one or more of the symptomsassociated with the cancer. As is understood in the clinical context, aneffective amount of a drug, compound, or pharmaceutical composition mayor may not be achieved in conjunction with another drug, compound, orpharmaceutical composition. Thus, an “effective amount” may beconsidered in the context of administering one or more therapeuticagents, and a single agent may be considered to be given in an effectiveamount if, in conjunction with one or more other agents, a desirableresult may be or is achieved.

As used herein, an “individual” or a “subject” refers to a mammal,including, but not limited to, human, bovine, horse, feline, canine,rodent, or primate. In some embodiments, the individual is a human.

The term “antibody” includes monoclonal antibodies (including fulllength 4-chain antibodies which have an immunoglobulin Fc region),antibody compositions with polyepitopic specificity, multispecificantibodies (e.g., bispecific antibodies, diabodies, and single-chainmolecules, as well as antibody fragments (e.g., Fab, F(ab′)₂, and Fv).The term “immunoglobulin” (Ig) is used interchangeably with “antibody”herein. Antibodies contemplated herein include heavy-chain onlyantibodies and sdAbs.

The basic 4-chain antibody unit is a heterotetrameric glycoproteincomposed of two identical light (L) chains and two identical heavy (H)chains. An IgM antibody consists of 5 of the basic heterotetramer unitsalong with an additional polypeptide called a J chain, and contains 10antigen binding sites, while IgA antibodies comprise from 2-5 of thebasic 4-chain units which can polymerize to form polyvalent assemblagesin combination with the J chain. In the case of IgGs, the 4-chain unitis generally about 150,000 daltons. Each L chain is linked to an H chainby one covalent disulfide bond, while the two H chains are linked toeach other by one or more disulfide bonds depending on the H chainisotype. Each H and L chain also has regularly spaced intrachaindisulfide bridges. Each H chain has at the N-terminus, a variable domain(V_(H)) followed by three constant domains (C_(H)) for each of the α andγ chains and four C_(H) domains for μ and ε isotypes. Each L chain hasat the N-terminus, a variable domain (V_(L)) followed by a constantdomain at its other end. The V_(L) is aligned with the V_(H) and theC_(L) is aligned with the first constant domain of the heavy chain(C_(H)1). Particular amino acid residues are believed to form aninterface between the light chain and heavy chain variable domains. Thepairing of a V_(H) and V_(L) together forms a single antigen-bindingsite. For the structure and properties of the different classes ofantibodies, see e.g., Basic and Clinical Immunology, 8th Edition, DanielP. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange,Norwalk, Conn., 1994, page 71 and Chapter 6. The L chain from anyvertebrate species can be assigned to one of two clearly distinct types,called kappa and lambda, based on the amino acid sequences of theirconstant domains. Depending on the amino acid sequence of the constantdomain of their heavy chains (C_(H)), immunoglobulins can be assigned todifferent classes or isotypes. There are five classes ofimmunoglobulins: IgA, IgD, IgE, IgG and IgM, having heavy chainsdesignated α, δ, ε, γ and μ, respectively. The γ and α classes arefurther divided into subclasses on the basis of relatively minordifferences in the C_(H) sequence and function, e.g., humans express thefollowing subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1 and IgA2.

An “isolated” antibody is one that has been identified, separated and/orrecovered from a component of its production environment (E.g., naturalor recombinant). Preferably, the isolated polypeptide is free ofassociation with all other components from its production environment.Contaminant components of its production environment, such as thatresulting from recombinant transfected cells, are materials that wouldtypically interfere with research, diagnostic or therapeutic uses forthe antibody, and may include enzymes, hormones, and other proteinaceousor non-proteinaceous solutes. In preferred embodiments, the polypeptidewill be purified: (1) to greater than 95% by weight of antibody asdetermined by, for example, the Lowry method, and in some embodiments,to greater than 99% by weight; (1) to a degree sufficient to obtain atleast 15 residues of N-terminal or internal amino acid sequence by useof a spinning cup sequenator, or (3) to homogeneity by SD S-PAGE undernon-reducing or reducing conditions using Coomassie blue or, preferably,silver stain. Isolated antibody includes the antibody in situ withinrecombinant cells since at least one component of the antibody's naturalenvironment will not be present. Ordinarily, however, an isolatedpolypeptide or antibody will be prepared by at least one purificationstep.

The “variable region” or “variable domain” of an antibody refers to theamino-terminal domains of the heavy or light chain of the antibody. Thevariable domains of the heavy chain and light chain may be referred toas “V_(H)” and “V_(L)”, respectively. These domains are generally themost variable parts of the antibody (relative to other antibodies of thesame class) and contain the antigen binding sites. Heavy-chain onlyantibodies from the Camelidae species have a single heavy chain variableregion, which is referred to as “V_(H)H”. V_(H)H is thus a special typeof V_(H).

The term “variable” refers to the fact that certain segments of thevariable domains differ extensively in sequence among antibodies. The Vdomain mediates antigen binding and defines the specificity of aparticular antibody for its particular antigen. However, the variabilityis not evenly distributed across the entire span of the variabledomains. Instead, it is concentrated in three segments calledhypervariable regions (HVRs) both in the light-chain and the heavy chainvariable domains. The more highly conserved portions of variable domainsare called the framework regions (FR). The variable domains of nativeheavy and light chains each comprise four FR regions, largely adopting abeta-sheet configuration, connected by three HVRs, which form loopsconnecting, and in some cases forming part of, the beta-sheet structure.The HVRs in each chain are held together in close proximity by the FRregions and, with the HVRs from the other chain, contribute to theformation of the antigen binding site of antibodies (see Kabat et al.,Sequences of Immunological Interest, Fifth Edition, National Instituteof Health, Bethesda, Md. (1991)). The constant domains are not involveddirectly in the binding of antibody to an antigen, but exhibit variouseffector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations and/orpost-translation modifications (e.g., isomerizations, amidations) thatmay be present in minor amounts. Monoclonal antibodies are highlyspecific, being directed against a single antigenic site. In contrast topolyclonal antibody preparations which typically include differentantibodies directed against different determinants (epitopes), eachmonoclonal antibody is directed against a single determinant on theantigen. In addition to their specificity, the monoclonal antibodies areadvantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The modifier “monoclonal”indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present application may be made by a variety of techniques,including, for example, the hybridoma method (e.g., Kohler andMilstein., Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14 (3):253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual, (ColdSpring Harbor Laboratory Press, 2^(nd) ed. 1988); Hammerling et al., in:Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y.,1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567),phage-display technologies (see, e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Sidhuet al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol.340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004), and technologies for producing human or human-likeantibodies in animals that have parts or all of the human immunoglobulinloci or genes encoding human immunoglobulin sequences (see, e.g., WO1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits etal., Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al.,Nature 362: 255-258 (1993); Bruggemann et al., Year in Immunol. 7:33(1993); U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126;5,633,425; and 5,661,016; Marks et al., Bio/Technology 10: 779-783(1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature368: 812-813 (1994); Fishwild et al., Nature Biotechnol. 14: 845-851(1996); Neuberger, Nature Biotechnol. 14: 826 (1996); and Lonberg andHuszar, Intern. Rev. Immunol. 13: 65-93 (1995).

The term “naked antibody” refers to an antibody that is not conjugatedto a cytotoxic moiety or radiolabel.

The terms “full-length antibody,” “intact antibody” or “whole antibody”are used interchangeably to refer to an antibody in its substantiallyintact form, as opposed to an antibody fragment. Specificallyfull-length 4-chain antibodies include those with heavy and light chainsincluding an Fc region. The constant domains may be native sequenceconstant domains (e.g., human native sequence constant domains) or aminoacid sequence variants thereof. In some cases, the intact antibody mayhave one or more effector functions.

An “antibody fragment” comprises a portion of an intact antibody,preferably the antigen binding and/or the variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂ andFv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870,Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]);single-chain antibody molecules and multispecific antibodies formed fromantibody fragments. Papain digestion of antibodies produced twoidentical antigen-binding fragments, called “Fab” fragments, and aresidual “Fc” fragment, a designation reflecting the ability tocrystallize readily. The Fab fragment consists of an entire L chainalong with the variable region domain of the H chain (V_(H)), and thefirst constant domain of one heavy chain (C_(H)1). Each Fab fragment ismonovalent with respect to antigen binding, i.e., it has a singleantigen-binding site. Pepsin treatment of an antibody yields a singlelarge F(ab′)₂ fragment which roughly corresponds to two disulfide linkedFab fragments having different antigen-binding activity and is stillcapable of cross-linking antigen. Fab′ fragments differ from Fabfragments by having a few additional residues at the carboxy terminus ofthe C_(H)1 domain including one or more cysteines from the antibodyhinge region. Fab′-SH is the designation herein for Fab′ in which thecysteine residue(s) of the constant domains bear a free thiol group.F(ab′)₂ antibody fragments originally were produced as pairs of Fab′fragments which have hinge cysteines between them. Other chemicalcouplings of antibody fragments are also known.

The Fc fragment comprises the carboxy-terminal portions of both H chainsheld together by disulfides. The effector functions of antibodies aredetermined by sequences in the Fc region, the region which is alsorecognized by Fc receptors (FcR) found on certain types of cells.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This fragment consists of a dimerof one heavy- and one light-chain variable region domain in tight,non-covalent association. From the folding of these two domains emanatesix hypervariable loops (3 loops each from the H and L chain) thatcontribute the amino acid residues for antigen binding and conferantigen binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three HVRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

“Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibodyfragments that comprise the V_(H) and V_(L) antibody domains connectedinto a single polypeptide chain. Preferably, the sFv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains whichenables the sFv to form the desired structure for antigen binding. For areview of the sFv, see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994).

“Functional fragments” of the antibodies described herein comprise aportion of an intact antibody, generally including the antigen bindingor variable region of the intact antibody or the Fc region of anantibody which retains or has modified FcR binding capability. Examplesof antibody fragments include linear antibody, single-chain antibodymolecules and multispecific antibodies formed from antibody fragments.

The term “diabodies” refers to small antibody fragments prepared byconstructing sFv fragments (see preceding paragraph) with short linkers(about 5-10) residues) between the V_(H) and V_(L) domains such thatinter-chain but not intra-chain pairing of the V domains is achieved,thereby resulting in a bivalent fragment, i.e., a fragment having twoantigen-binding sites. Bispecific diabodies are heterodimers of two“crossover” sFv fragments in which the V_(H) and V_(L) domains of thetwo antibodies are present on different polypeptide chains. Diabodiesare described in greater detail in, for example, EP 404,097; WO93/11161; Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448(1993).

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is(are) identical with or homologous to corresponding sequencesin antibodies derived from another species or belonging to anotherantibody class or subclass, as well as fragments of such antibodies, solong as they exhibit the desired biological activity (U.S. Pat. No.4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855(1984)). Chimeric antibodies of interest herein include PRIMATTZFD®antibodies wherein the antigen-binding region of the antibody is derivedfrom an antibody produced by, e.g., immunizing macaque monkeys with anantigen of interest. As used herein, “humanized antibody” is used asubset of “chimeric antibodies.”

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. In one embodiment, a humanized antibody is a humanimmunoglobulin (recipient antibody) in which residues from an HVR(hereinafter defined) of the recipient are replaced by residues from anHVR of a non-human species (donor antibody) such as mouse, rat, rabbitor non-human primate having the desired specificity, affinity, and/orcapacity. In some instances, framework (“FR”) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues that are notfound in the recipient antibody or in the donor antibody. Thesemodifications may be made to further refine antibody performance, suchas binding affinity. In general, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin sequence, and all orsubstantially all of the FR regions are those of a human immunoglobulinsequence, although the FR regions may include one or more individual FRresidue substitutions that improve antibody performance, such as bindingaffinity, isomerization, immunogenicity, etc. The number of these aminoacid substitutions in the FR is typically no more than 6 in the H chain,and in the L chain, no more than 3. The humanized antibody optionallywill also comprise at least a portion of an immunoglobulin constantregion (Fc), typically that of a human immunoglobulin. For furtherdetails, see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmannet al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.2:593-596 (1992). See also, for example, Vaswani and Hamilton, Ann.Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc.Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech.5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and 7,087,409.

A “human antibody” is an antibody that possesses an amino-acid sequencecorresponding to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues. Human antibodies can be produced using various techniquesknown in the art, including phage-display libraries. Hoogenboom andWinter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol.,222:581 (1991). Also available for the preparation of human monoclonalantibodies are methods described in Cole et al., Monoclonal Antibodiesand Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J.Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel,Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can beprepared by administering the antigen to a transgenic animal that hasbeen modified to produce such antibodies in response to antigenicchallenge, but whose endogenous loci have been disabled, e.g., immunizedxenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regardingXENOMOUSE™ technology). See also, for example, Li et al., Proc. Natl.Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodiesgenerated via a human B-cell hybridoma technology.

The term “hypervariable region,” “HVR,” or “HV,” when used herein refersto the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops. Generally, 4-chainantibodies comprise six HVRs; three in the V_(H) (H1, H2, H3), and threein the V_(L) (L1, L2, L3). Single-domain antibodies comprise three HVRs,such as three in the V_(H)H (H1, H2, H3). In native 4-chain antibodies,H3 and L3 display the most diversity of the six HVRs, and H3 inparticular is believed to play a unique role in conferring finespecificity to antibodies. See, e.g., Xu et al., Immunity 13:37-45(2000); Johnson and Wu, in Methods in Molecular Biology 248:1-25 (Lo,ed., Human Press, Totowa, N.J., 2003). Indeed, naturally occurringcamelid antibodies consisting of a heavy chain only are functional andstable in the absence of light chain. See, e.g., Hamers-Casterman etal., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol.3:733-736 (1996).

The term “Complementarity Determining Region” or “CDR” are used to referto hypervariable regions as defined by the Kabat system. See Kabat etal., Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991)

A number of HVR delineations are in use and are encompassed herein. TheKabat Complementarity Determining Regions (CDRs) are based on sequencevariability and are the most commonly used (Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). Chothia refersinstead to the location of the structural loops (Chothia and Lesk, J.Mol. Biol. 196:901-917 (1987)). The AbM HVRs represent a compromisebetween the Kabat HVRs and Chothia structural loops, and are used byOxford Molecular's AbM antibody modeling software. The “contact” HVRsare based on an analysis of the available complex crystal structures.The residues from each of these HVRs are noted below in Table I.

TABLE I HVR delineations. Loop Kabat AbM Chothia Contact L1 L24-L34L24-L34 L26-L32 L30-L36 L2 L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97L89-L97 L91-L96 L89-L96 H1 H31-H35B H26-H35B H26-H32 H30-H35B (KabatNumbering) H1 H31-H35 H26-H35 H26-H32 H30-H35 (Chothia Numbering) H2H50-H65 H50-H58 H53-H55 H47-H58 H3 H95-H102 H95-H102 H96-H101 H93-H101

HVRs may comprise “extended HVRs” as follows: 24-36 or 24-34 (L1), 46-56or 50-56 (L2) and 89-97 or 89-96 (L3) in the V_(L) and 26-35 (H1), 50-65or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in the V_(H). Thevariable domain residues are numbered according to Kabat et al., supra,for each of these definitions.

The expression “variable-domain residue-numbering as in Kabat” or“amino-acid-position numbering as in Kabat,” and variations thereof,refers to the numbering system used for heavy-chain variable domains orlight-chain variable domains of the compilation of antibodies in Kabatet al., supra. Using this numbering system, the actual linear amino acidsequence may contain fewer or additional amino acids corresponding to ashortening of, or insertion into, a FR or HVR of the variable domain.For example, a heavy-chain variable domain may include a single aminoacid insert (residue 52a according to Kabat) after residue 52 of H2 andinserted residues (e.g. residues 82a, 82b, and 82c, etc. according toKabat) after heavy-chain FR residue 82. The Kabat numbering of residuesmay be determined for a given antibody by alignment at regions ofhomology of the sequence of the antibody with a “standard” Kabatnumbered sequence.

“Framework” or “FR” residues are those variable-domain residues otherthan the HVR residues as herein defined.

A “human consensus framework” or “acceptor human framework” is aframework that represents the most commonly occurring amino acidresidues in a selection of human immunoglobulin V_(L) or V_(H) frameworksequences. Generally, the selection of human immunoglobulin V_(L) orV_(H) sequences is from a subgroup of variable domain sequences.Generally, the subgroup of sequences is a subgroup as in Kabat et al.,Sequences of Proteins of Immunological Interest, 5^(th) Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991).Examples include for the V_(L), the subgroup may be subgroup kappa I,kappa II, kappa III or kappa IV as in Kabat et al., supra. Additionally,for the VH, the subgroup may be subgroup I, subgroup II, or subgroup IIIas in Kabat et al. Alternatively, a human consensus framework can bederived from the above in which particular residues, such as when ahuman framework residue is selected based on its homology to the donorframework by aligning the donor framework sequence with a collection ofvarious human framework sequences. An acceptor human framework “derivedfrom” a human immunoglobulin framework or a human consensus frameworkmay comprise the same amino acid sequence thereof, or it may containpre-existing amino acid sequence changes. In some embodiments, thenumber of pre-existing amino acid changes are 10 or less, 9 or less, 8or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 orless.

An “amino-acid modification” at a specified position, e.g. of the Fcregion, refers to the substitution or deletion of the specified residue,or the insertion of at least one amino acid residue adjacent thespecified residue. Insertion “adjacent” to a specified residue meansinsertion within one to two residues thereof. The insertion may beN-terminal or C-terminal to the specified residue. The preferred aminoacid modification herein is a substitution.

An “affinity-matured” antibody is one with one or more alterations inone or more HVRs thereof that result in an improvement in the affinityof the antibody for antigen, compared to a parent antibody that does notpossess those alteration(s). In one embodiment, an affinity-maturedantibody has nanomolar or even picomolar affinities for the targetantigen. Affinity-matured antibodies are produced by procedures known inthe art. For example, Marks et al., Bio/Technology 10:779-783 (1992)describes affinity maturation by V_(H)- and V_(L)-domain shuffling.Random mutagenesis of HVR and/or framework residues is described by, forexample: Barbas et al. Proc Nat. Acad. Sci. USA 91:3809-3813 (1994);Schier et al. Gene 169:147-155 (1995); Yelton et al. J. Immunol.155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995);and Hawkins et al, J. Mol. Biol. 226:889-896 (1992).

As use herein, the term “specifically binds” or is “specific for” refersto measurable and reproducible interactions such as binding between atarget and an antibody, which is determinative of the presence of thetarget in the presence of a heterogeneous population of moleculesincluding biological molecules. For example, an antibody thatspecifically binds a target (which can be an epitope) is an antibodythat binds this target with greater affinity, avidity, more readily,and/or with greater duration than it binds other targets. In oneembodiment, the extent of binding of an antibody to an unrelated targetis less than about 10% of the binding of the antibody to the target asmeasured, e.g., by a radioimmunoassay (RIA). In certain embodiments, anantibody that specifically binds a target has a dissociation constant(Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, or X0.1 nM. In certainembodiments, an antibody specifically binds an epitope on a protein thatis conserved among the protein from different species. In anotherembodiment, specific binding can include, but does not require exclusivebinding.

The term “specificity” refers to selective recognition of an antigenbinding protein or antibody for a particular epitope of an antigen.Natural antibodies, for example, are monospecific. The term“multispecific” as used herein denotes that an antigen binding proteinor an antibody has two or more antigen-binding sites of which at leasttwo bind a different antigen or a different epitope of the same antigen.“Bispecific” as used herein denotes that an antigen binding protein oran antibody has two different antigen-binding specificities. The term“monospecific” antibody as used herein denotes an antibody that has oneor more binding sites each of which bind the same epitope of the sameantigen.

The term “valent” as used herein denotes the presence of a specifiednumber of binding sites in an antigen binding protein or antibodymolecule. A natural antibody for example or a full length antibody hastwo binding sites and is bivalent. As such, the terms “trivalent”,“tetravalent”, “pentavalent” and “hexavalent” denote the presence of twobinding site, three binding sites, four binding sites, five bindingsites, and six binding sites, respectively, in an antigen bindingprotein or antibody molecule. The MABPs of the present application areat least “bivalent,” for example, the MABPs can be “trivalent,” or“tetravalent.”

A “blocking” antibody or an “antagonist” antibody is one that inhibitsor reduces a biological activity of the antigen it binds. In someembodiments, blocking antibodies or antagonist antibodies substantiallyor completely inhibit the biological activity of the antigen.

An “agonist” or activating antibody is one that enhances or initiatessignaling by the antigen to which it binds. In some embodiments, agonistantibodies cause or activate signaling without the presence of thenatural ligand.

“Antibody effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody, and vary with the antibodyisotype. Examples of antibody effector functions include: C1q bindingand complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and Bcell activation. “Reduced or minimized” antibody effector function meansthat which is reduced by at least 50% (alternatively 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) from the wild type or unmodifiedantibody. The determination of antibody effector function is readilydeterminable and measurable by one of ordinary skill in the art. In apreferred embodiment, the antibody effector functions of complementbinding, complement dependent cytotoxicity and antibody dependentcytotoxicity are affected. In some embodiments, effector function iseliminated through a mutation in the constant region that eliminatedglycosylation, e.g., “effector-less mutation.” In one aspect, theeffector-less mutation is an N297A or DANA mutation (D265A+N297A) in theC_(H)2 region. Shields et al., J. Biol. Chem. 276 (9): 6591-6604 (2001).Alternatively, additional mutations resulting in reduced or eliminatedeffector function include: K322A and L234A/L235A (LALA). Alternatively,effector function can be reduced or eliminated through productiontechniques, such as expression in host cells that do not glycosylate(e.g., E. coli.) or in which result in an altered glycosylation patternthat is ineffective or less effective at promoting effector function(e.g., Shinkawa et al., J. Biol. Chem. 278(5): 3466-3473 (2003).

“Antibody-dependent cell-mediated cytotoxicity” or ADCC refers to a formof cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)present on certain cytotoxic cells (e.g., natural killer (NK) cells,neutrophils and macrophages) enable these cytotoxic effector cells tobind specifically to an antigen-bearing target cell and subsequentlykill the target cell with cytotoxins. The antibodies “arm” the cytotoxiccells and are required for killing of the target cell by this mechanism.The primary cells for mediating ADCC, NK cells, express FcγRIII only,whereas monocytes express FcγRI, FcγRII and FcγRIII Fc expression onhematopoietic cells is summarized in Table 3 on page 464 of Ravetch andKinet, Annu. Rev. Immunol. 9: 457-92 (1991). To assess ADCC activity ofa molecule of interest, an in vitro ADCC assay, such as that describedin U.S. Pat. No. 5,500,362 or 5,821,337 may be performed. Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and natural killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g., in an animal model such as that disclosed in Clynes etal., PNAS USA 95:652-656 (1998).

Unless indicated otherwise herein, the numbering of the residues in animmunoglobulin heavy chain is that of the EU index as in Kabat et al.,supra. The “EU index as in Kabat” refers to the residue numbering of thehuman IgG1 EU antibody.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain, including native-sequence Fc regions andvariant Fc regions. Although the boundaries of the Fc region of animmunoglobulin heavy chain might vary, the human IgG heavy-chain Fcregion is usually defined to stretch from an amino acid residue atposition Cys226, or from Pro230, to the carboxyl-terminus thereof. TheC-terminal lysine (residue 447 according to the EU numbering system) ofthe Fc region may be removed, for example, during production orpurification of the antibody, or by recombinantly engineering thenucleic acid encoding a heavy chain of the antibody. Accordingly, acomposition of intact antibodies may comprise antibody populations withall K447 residues removed, antibody populations with no K447 residuesremoved, and antibody populations having a mixture of antibodies withand without the K447 residue. Suitable native-sequence Fc regions foruse in the antibodies described herein include human IgG1, IgG2 (IgG2A,IgG2B), IgG3 and IgG4.

“Fc receptor” or “FcR” describes a receptor that binds the Fc region ofan antibody. The preferred FcR is a native sequence human FcR. Moreover,a preferred FcR is one which binds an IgG antibody (a gamma receptor)and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses,including allelic variants and alternatively spliced forms of thesereceptors, FcγRII receptors include FcγRIIA (an “activating receptor”)and FcγRIIB (an “inhibiting receptor”), which have similar amino acidsequences that differ primarily in the cytoplasmic domains thereof.Activating receptor FcγRIIA contains an immunoreceptor tyrosine-basedactivation motif (ITAM) in its cytoplasmic domain. Inhibiting receptorFcγRIIB contains an immunoreceptor tyrosine-based inhibition motif(ITIM) in its cytoplasmic domain. (see M. Daëron, Annu. Rev. Immunol.15:203-234 (1997). FcRs are reviewed in Ravetch and Kinet, Annu. Rev.Immunol. 9: 457-92 (1991); Capel et al., Immunomethods 4: 25-34 (1994);and de Haas et al., J. Lab. Clin. Med. 126: 330-41 (1995). Other FcRs,including those to be identified in the future, are encompassed by theterm “FcR” herein.

The term “Fc receptor” or “FcR” also includes the neonatal receptor,FcRn, which is responsible for the transfer of maternal IgGs to thefetus. Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J.Immunol. 24: 249 (1994). Methods of measuring binding to FcRn are known(see, e.g., Ghetie and Ward, Immunol. Today 18: (12): 592-8 (1997);Ghetie et al., Nature Biotechnology 15 (7): 637-40 (1997); Hinton etal., J. Biol. Chem. 279 (8): 6213-6 (2004); WO 2004/92219 (Hinton etal.). Binding to FcRn in vivo and serum half-life of human FcRnhigh-affinity binding polypeptides can be assayed, e.g., in transgenicmice or transfected human cell lines expressing human FcRn, or inprimates to which the polypeptides having a variant Fc region areadministered. WO 2004/42072 (Presta) describes antibody variants whichimproved or diminished binding to FcRs. See also, e.g., Shields et al.,J. Biol. Chem. 9(2): 6591-6604 (2001).

“Effector cells” are leukocytes which express one or more FcRs andperform effector functions. In one aspect, the effector cells express atleast FcγRIII and perform ADCC effector function. Examples of humanleukocytes which mediate ADCC include peripheral blood mononuclear cells(PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells andneutrophils. The effector cells may be isolated from a native source,e.g., blood. Effector cells generally are lymphocytes associated withthe effector phase, and function to produce cytokines (helper T cells),killing cells in infected with pathogens (cytotoxic T cells) orsecreting antibodies (differentiated B cells).

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (C1q) to antibodies (of the appropriate subclass)which are bound to their cognate antigen. To assess complementactivation, a CDC assay, e.g., as described in Gazzano-Santoro et al.,J. Immunol. Methods 202: 163 (1996), may be performed. Antibody variantswith altered Fc region amino acid sequences and increased or decreasedC1q binding capability are described in U.S. Pat. No. 6,194,551B1 andWO99/51642. The contents of those patent publications are specificallyincorporated herein by reference. See, also, Idusogie et al. J. Immunol.164: 4178-4184 (2000).

The term “heavy chain-only antibody” or “HCAb” refers to a functionalantibody, which comprises heavy chains, but lacks the light chainsusually found in antibodies. Camelid animals (such as camels, llamas, oralpacas) are known to produce HCAbs.

The term “single-domain antibody” or “sdAb” refers to a singleantigen-binding polypeptide having three complementary determiningregions (CDRs). The sdAb alone is capable of binding to the antigenwithout pairing with a corresponding CDR-containing polypeptide. In somecases, sdAbs are engineered from camelid HCAbs, and their heavy chainvariable domains are referred herein as “V_(H)Hs”. Camelid sdAb is oneof the smallest known antigen-binding antibody fragments (see, e.g.,Hamers-Casterman et al., Nature 363:446-8 (1993); Greenberg et al.,Nature 374:168-73 (1995); Hassanzadeh-Ghassabeh et al., Nanomedicine(Lond), 8:1013-26 (2013)).

“Binding affinity” generally refers to the strength of the sum total ofnon-covalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity that reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (Kd). Affinity can be measured by common methodsknown in the art, including those described herein. Low-affinityantibodies generally bind antigen slowly and tend to dissociate readily,whereas high-affinity antibodies generally bind antigen faster and tendto remain bound longer. A variety of methods of measuring bindingaffinity are known in the art, any of which can be used for purposes ofthe present application. Specific illustrative and exemplary embodimentsfor measuring binding affinity are described in the following.

The “Kd” or “Kd value” as used herein is in one embodiment measured by aradiolabeled antigen binding assay (RIA) performed with the Fab versionof the antibody and antigen molecule as described by the following assaythat measures solution binding affinity of Fabs for antigen byequilibrating Fab with a minimal concentration of (¹²⁵I)-labeled antigenin the presence of a titration series of unlabeled antigen, thencapturing bound antigen with an anti-Fab antibody-coated plate (Chen, etal., (1999) J. Mol. Biol 293:865-881). To establish conditions for theassay, microtiter plates (Dynex) are coated overnight with 5 μg/ml of acapturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH9.6), and subsequently blocked with 2% (w/v) bovine serum albumin in PBSfor two to five hours at room temperature (approximately 23° C.). In anon-adsorbent plate (Nunc #269620), 100 pM or 26 pM [¹²⁵I]-antigen aremixed with serial dilutions of a Fab of interest (consistent withassessment of an anti-VEGF antibody, Fab-12, in Presta et al., (1997)Cancer Res. 57:4593-4599). The Fab of interest is then incubatedovernight; however, the incubation may continue for a longer period(e.g., 65 hours) to insure that equilibrium is reached. Thereafter, themixtures are transferred to the capture plate for incubation at roomtemperature for one hour. The solution is then removed and the platewashed eight times with 0.1% Tween-20 in PBS. When the plates havedried, 150 μl/well of scintillant (MicroScint-20; Packard) is added, andthe plates are counted on a Topcount gamma counter (Packard) for tenminutes. Concentrations of each Fab that give less than or equal to 20%of maximal binding are chosen for use in competitive binding assays.

According to another embodiment, the Kd is measured by usingsurface-plasmon resonance assays using a BIACORE®-2000 or aBIACORE®-3000 instrument (BIAcore, Inc., Piscataway, N.J.) at 25° C.with immobilized antigen CMS chips at ^(˜)10 response units (RU).Briefly, carboxymethylated dextran biosensor chips (CMS, BIAcore Inc.)are activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Antigen is diluted with 10 mM sodium acetate,pH 4.8, to 5 μg/ml C0.2 μM) before injection at a flow rate of 5μL/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of antigen, 1 M ethanolamine isinjected to block unreacted groups. For kinetics measurements, two-foldserial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with0.05% TWEEN 20™ surfactant (PBST) at 25° C. at a flow rate ofapproximately 25 μL/min. Association rates (k_(on)) and dissociationrates (k_(off)) are calculated using a simple one-to-one Langmuirbinding model (BIAcore® Evaluation Software version 3.2) bysimultaneously fitting the association and dissociation sensorgrams. Theequilibrium dissociation constant (Kd) is calculated as the ratiok_(off)/k_(on). See, e.g., Chen et al., J. Mol. Biol. 293:865-881(1999). If the on-rate exceeds 10⁶M⁻¹s⁻¹ by the surface-plasmonresonance assay above, then the on-rate can be determined by using afluorescent quenching technique that measures the increase or decreasein fluorescence-emission intensity (excitation=295 nm; emission=340 nm,16 nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab form)in PBS, pH 7.2, in the presence of increasing concentrations of antigenas measured in a spectrometer, such as a stop-flow-equippedspectrophotometer (Aviv Instruments) or a 8000-series SLM-AMINCO™spectrophotometer (ThermoSpectronic) with a stirred cuvette.

An “on-rate,” “rate of association,” “association rate,” or “k_(on)” asused herein can also be determined as described above using aBIACORE®-2000 or a BIACORE®-3000 system (BIAcore, Inc., Piscataway,N.J.) at 25° C. with immobilized antigen CMS chips at about 10 responseunits (RU). Briefly, carboxymethylated dextran biosensor ships (CMS,BIAcore Inc.) are activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (ECD) and N-hydroxysuccinimide (NHS)according to the supplier's instructions. Antigen is diluted with 10 mMsodium acetate, pH 4.8, into 5 mg/ml 0.2 mM) before injection at a flowrate of 5 ml/min. to achieve approximately 10 response units (RU) ofcoupled protein. Following the injection of antigen, 1M ethanolamine isadded to block unreacted groups. For kinetics measurements, two-foldserial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with0.05% Tween 20 (PBST) at 25° C. at a flow rate of approximately 25μl/min. Association rates (k_(on)) and dissociation rates (k_(off)) arecalculated using a simple one-to-one Langmuir binding model (BIAcoreEvaluation Software version 3.2) by simultaneous fitting the associationand dissociation sensorgram. The equilibrium dissociation constant (Kd)was calculated as the ratio k_(off)/k_(on). See, e.g., Chen, Y., et al.,(1999) J. Mol. Biol 293:865-881. However, if the on-rate exceeds 10⁶M⁻¹S⁻¹ by the surface plasmon resonance assay above, then the on-rate ispreferably determined by using a fluorescent quenching technique thatmeasures the increase or decrease in fluorescence emission intensity(excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence ofincreasing concentrations of antigen as measured in a spectrometer, suchas a stop-flow equipped spectrophometer (Aviv Instruments) or a8000-series SLM-Aminco spectrophotometer (ThermoSpectronic) with astirred cuvette.

“Percent (%) amino acid sequence identity” and “homology” with respectto a peptide, polypeptide or antibody sequence are defined as thepercentage of amino acid residues in a candidate sequence that areidentical with the amino acid residues in the specific peptide orpolypeptide sequence, after aligning the sequences and introducing gaps,if necessary, to achieve the maximum percent sequence identity, and notconsidering any conservative substitutions as part of the sequenceidentity. Alignment for purposes of determining percent amino acidsequence identity can be achieved in various ways that are within theskill in the art, for instance, using publicly available computersoftware such as BLAST, BLAST-2, ALIGN or MEGALIGN™ (DNASTAR) software.Those skilled in the art can determine appropriate parameters formeasuring alignment, including any algorithms needed to achieve maximalalignment over the full length of the sequences being compared.

An “isolated” nucleic acid molecule encoding the MABP or sdAb herein isa nucleic acid molecule that is identified and separated from at leastone contaminant nucleic acid molecule with which it is ordinarilyassociated in the environment in which it was produced. Preferably, theisolated nucleic acid is free of association with all componentsassociated with the production environment. The isolated nucleic acidmolecules encoding the polypeptides and antibodies herein is in a formother than in the form or setting in which it is found in nature.Isolated nucleic acid molecules therefore are distinguished from nucleicacid encoding the polypeptides and antibodies herein existing naturallyin cells.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers that are nontoxic to the cell or mammal beingexposed thereto at the dosages and concentrations employed. Often thephysiologically acceptable carrier is an aqueous pH buffered solution.Examples of physiologically acceptable carriers include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptide; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugars such as sucrose, mannitol, trehalose orsorbitol; salt-forming counterions such as sodium; metal complexes (e.g.Zn-protein complexes); and/or nonionic surfactants such as TWEEN™,polyethylene glycol (PEG), and PLURONICS™ or polyethylene glycol (PEG).

The “diluent” of interest herein is one which is pharmaceuticallyacceptable (safe and non-toxic for administration to a human) and isuseful for the preparation of a liquid formulation, such as aformulation reconstituted after lyophilization. Exemplary diluentsinclude sterile water, bacteriostatic water for injection (BWFI), a pHbuffered solution (e.g. phosphate-buffered saline), sterile salinesolution, Ringer's solution or dextrose solution. In an alternativeembodiment, diluents can include aqueous solutions of salts and/orbuffers.

A “preservative” is a compound which can be added to the formulationsherein to reduce bacterial activity. The addition of a preservative may,for example, facilitate the production of a multi-use (multiple-dose)formulation. Examples of potential preservatives includeoctadecyldimethylbenzyl ammonium chloride, hexamethonium chloride,benzalkonium chloride (a mixture of alkylbenzyldimethylammoniumchlorides in which the alkyl groups are long-chain compounds), andbenzethonium chloride. Other types of preservatives include aromaticalcohols such as phenol, butyl and benzyl alcohol, alkyl parabens suchas methyl or propyl paraben, catechol, resorcinol, cyclohexanol,3-pentanol, and m-cresol. The most preferred preservative herein isbenzyl alcohol.

The term “pharmaceutical formulation” refers to a preparation that is insuch form as to permit the biological activity of the active ingredientto be effective, and that contains no additional components that areunacceptably toxic to a subject to which the formulation would beadministered. Such formulations are sterile. A “sterile” formulation isaseptic or free from all living microorganisms and their spores.

A “stable” formulation is one in which the protein therein essentiallyretains its physical and chemical stability and integrity upon storage.Various analytical techniques for measuring protein stability areavailable in the art and are reviewed in Peptide and Protein DrugDelivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y.,Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993).Stability can be measured at a selected temperature for a selected timeperiod. For rapid screening, the formulation may be kept at 40° C. for 2weeks to 1 month, at which time stability is measured. Where theformulation is to be stored at 2-8° C., generally the formulation shouldbe stable at 30° C. or 40° C. for at least 1 month and/or stable at 2-8°C. for at least 2 years. Where the formulation is to be stored at 30°C., generally the formulation should be stable for at least 2 years at30° C. and/or stable at 40° C. for at least 6 months. For example, theextent of aggregation during storage can be used as an indicator ofprotein stability. Thus, a “stable” formulation may be one wherein lessthan about 10% and preferably less than about 5% of the protein arepresent as an aggregate in the formulation. In other embodiments, anyincrease in aggregate formation during storage of the formulation can bedetermined.

A “reconstituted” formulation is one which has been prepared bydissolving a lyophilized protein or antibody formulation in a diluentsuch that the protein is dispersed throughout. The reconstitutedformulation is suitable for administration (e.g. subcutaneousadministration) to a patient to be treated with the protein of interestand, in certain embodiments, may be one which is suitable for parenteralor intravenous administration.

An “isotonic” formulation is one which has essentially the same osmoticpressure as human blood. Isotonic formulations will generally have anosmotic pressure from about 250 to 350 mOsm. The term “hypotonic”describes a formulation with an osmotic pressure below that of humanblood. Correspondingly, the term “hypertonic” is used to describe aformulation with an osmotic pressure above that of human blood.Isotonicity can be measured using a vapor pressure or ice-freezing typeosmometer, for example. The formulations of the present application canbe hypertonic as a result of the addition of salt and/or buffer.

“Immune checkpoint molecules” refers to molecules in the immune systemthat either turn up a signal or turn down a signal. “Stimulatory immunecheckpoint molecules” or “co-stimulatory molecules” are immunecheckpoint molecules that turn up a signal in the immune system.“Inhibitory immune checkpoint molecules” are immune checkpoint moleculesthat turn down a signal in the immune system.

It is understood that embodiments described herein include “consisting”and/or “consisting essentially of” embodiments.

Reference to “about” a value or parameter herein includes (anddescribes) variations that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X”.

As used herein, reference to “not” a value or parameter generally meansand describes “other than” a value or parameter. For example, the methodis not used to treat cancer of type X means the method is used to treatcancer of types other than X.

The term “about X-Y” used herein has the same meaning as “about X toabout Y.”

As used herein and in the appended claims, the singular forms “a,” “or,”and “the” include plural referents unless the context clearly dictatesotherwise.

II. Multispecific Antigen Binding Proteins (MABPs)

One aspect of the present application provides a multispecific antigenbinding protein (MABP) comprising: (a) a first antigen binding portioncomprising a heavy chain variable domain (V_(H)) and a light chainvariable domain (V_(L)), wherein the V_(H) and V_(L) together form anantigen-binding site that specifically binds a first epitope, and (b) asecond antigen binding portion comprising an sdAb that specificallybinds a second epitope, wherein the first antigen binding portion andthe second antigen binding portion are fused to each other. In someembodiments, the first epitope is from a first immune checkpointmolecule, and the second epitope is from a second immune checkpointmolecule. In some embodiments, the first epitope is from a first tumorantigen, and the second epitope is from a second tumor antigen. In someembodiments, the first epitope is from a tumor antigen, and the secondepitope is from a cell surface molecule, such as CD3. In someembodiments, the sdAb is a camelid, humanized, or human sdAb. In someembodiments, the first epitope is from a first pro-inflammatorymolecule, and the second epitope is from a second pro-inflammatorymolecule. In some embodiments, the first antigen binding portioncomprises a heavy chain comprising the V_(H) and a light chaincomprising the V_(L). In some embodiments, the second antigen bindingportion is fused to the first antigen binding portion at the N-terminusof the heavy chain, the N-terminus of the light chain, the N-terminus ofthe Fc region, the C-terminus of the heavy chain, or the C-terminus ofthe light chain. In some embodiments, the first antigen binding portioncomprises a full-length 4-chain antibody. In some embodiments, thesecond antigen binding portion is fused to the first antigen bindingportion chemically. In some embodiments, the second antigen bindingportion is fused to the first antigen binding portion via a peptide bondor a peptide linker. In some embodiments, the peptide linker is no morethan about 30 (such as no more than about any one of 25, 20, or 15)amino acids long. In some embodiments, the first antigen bindingfragment comprises an Fc region, such as an IgG1 Fc or IgG4 Fc.

The MABPs of the present application have at least two antigen bindingportions that can specifically bind at least two different epitopes.Some of the at least two antigen binding portions may be identical, solong as the MABP has binding sites for two different epitopes. The MABPscan be symmetric or asymmetric. For example, the MABP may comprise oneor two copies of the first antigen binding portion, and one to eightcopies of the second antigen binding portion. In some embodiments, theMABP comprises two different antigen binding portions that each comprisea V_(H) domain and a V_(L) domain that together form a different antigenbinding site. For example, the first antigen binding portion can be abispecific antibody. In some embodiments, the first antigen bindingportion is a monospecific full-length antibody or antigen bindingfragment thereof, such as a Fab.

In some embodiments, the MABP comprises any one of 1, 2, 3, 4, 5, 6, 7,8, or more different antigen binding portions that each comprises ansdAb. In some embodiments, two identical sdAbs are fused to each other,which is further fused to the first antigen binding portion. In someembodiments, two different sdAbs are fused to each other, which isfurther fused to the first antigen binding portion.

The MABPs may have any suitable number of valencies for each epitope,and any suitable number of specificity. In some embodiments, the MABP isbivalent, trivalent, tetravalent, pentavalent, hexavalent, or of highervalencies for the first epitope. In some embodiments, the MABP isbivalent, trivalent, tetravalent, pentavalent, hexavalent, or of highervalencies for the second epitope. In some embodiments, the MABP isbispecific. In some embodiments, the MABP is trispecific. In someembodiments, the MABP is tetraspecific. In some embodiments, the MABPhas more than four specificities. Exemplary MABPs are depicted in FIGS.1-22 .

In some embodiments, there is provided a bispecific antigen bindingprotein (“BABP”) comprising: (a) a single copy of a first antigenbinding portion comprising a heavy chain variable domain (V_(H)) and alight chain variable domain (V_(L)), wherein the V_(H) and V_(L)together form an antigen-binding site that specifically binds a firstepitope, and (b) one or more copies (such as 2) of a second antigenbinding portion comprising an sdAb that specifically binds a secondepitope, wherein each copy of the second antigen binding portion isfused to the first antigen binding portion. An example is shown in FIG.5 . In some embodiments, one or more of the sdAbs is each further fusedto another identical or different sdAb.

In some embodiments, there is provided a MABP comprising: (a) aplurality (such as 2, 3, 4, 5, 6, or more) of a first antigen bindingportion comprising a heavy chain variable domain (V_(H)) and a lightchain variable domain (V_(L)), wherein the V_(H) and V_(L) together forman antigen-binding site that specifically binds a first epitope, and (b)a plurality (such as 2, 3, 4, 5, 6, 7, 8, or more) of identical ordifferent sdAbs that each specifically binds an epitope that isdifferent from the first epitope, wherein the sdAbs are fused to eachother, and/or to the first antigen binding portion.

In some embodiments, there is provided a multispecific (such asbispecific) antigen binding protein comprising: (a) two copies of afirst antigen binding portion each comprising a heavy chain variabledomain (V_(H)) and a light chain variable domain (V_(L)), wherein theV_(H) and V_(L) together form an antigen-binding site that specificallybinds a first epitope, and (b) a single copy of a second antigen bindingportion comprising an sdAb that specifically binds a second epitope,wherein the second antigen binding portion is fused to one of the twocopies of the first antigen binding portion. An example is shown in FIG.1 .

In some embodiments, there is provided a multispecific (such asbispecific) antigen binding protein comprising: (a) two copies of afirst antigen binding portion each comprising a heavy chain variabledomain (V_(H)) and a light chain variable domain (V_(L)), wherein theV_(H) and V_(L) together form an antigen-binding site that specificallybinds a first epitope, and (b) a plurality (such as 2, 3, or 4) ofidentical or different sdAbs that each specifically binds an epitopethat is different from the first epitope, wherein the sdAbs are fused toeach other, and/or to the first antigen binding portion. Examples areshown in FIGS. 2, 3, 17, 18, 21, and 22 .

In some embodiments, there is provided a multispecific (such asbispecific) antigen binding protein comprising: (a) two copies of afirst antigen binding portion each comprising a heavy chain variabledomain (V_(H)) and a light chain variable domain (V_(L)), wherein theV_(H) and V_(L) together form an antigen-binding site that specificallybinds a first epitope, and (b) two copies of a second antigen bindingportion each comprising an sdAb that specifically binds a secondepitope, wherein one copy of the second antigen binding portion is fusedto each copy of the first antigen binding portion. Examples are shown inFIGS. 4, 9, 11, 13, 19, and 20 . In some embodiments, one or more of thesdAbs is each further fused to another identical or different sdAb.

In some embodiments, there is provided a multispecific (such astrispecific) antigen binding protein comprising: (a) a first copy and asecond copy of a first antigen binding portion each comprising a heavychain variable domain (V_(H)) and a light chain variable domain (V_(L)),wherein the V_(H) and V_(L) together form an antigen-binding site thatspecifically binds a first epitope, (b) a second antigen binding portioncomprising an sdAb that specifically binds a second epitope, and (c) athird antigen binding portion comprising a second sdAb that specificallybinds a third epitope, wherein the second antigen binding portion isfused to the first copy of the first antigen binding portion, andwherein the third antigen binding portion is fused to the second copy ofthe first antigen binding portion. Examples are shown in FIGS. 7, 10, 12, and 14. In some embodiments, one or more of the sdAbs is each furtherfused to another identical or different sdAb.

In some embodiments, there is provided a multispecific (such astrispecific) antigen binding protein comprising: (a) a first antigenbinding portion comprising a first heavy chain variable domain (V_(H))and a first light chain variable domain (V_(L)), wherein the first V_(H)and first V_(L) together form a first antigen-binding site thatspecifically binds a first epitope; (b) one to four copies of a secondantigen binding portion comprising an sdAb that specifically binds asecond epitope; and (c) a third antigen binding portion comprising athird heavy chain variable domain (V_(H)) and a third light chainvariable domain (V_(L)), wherein the third V_(H) and third V_(L)together form a third antigen-binding site that specifically binds athird epitope; and wherein the second antigen binding portion is fusedto the first antigen binding portion and/or the third antigen bindingportion. An example is shown in FIG. 6 . In some embodiments, one ormore of the sdAbs is each further fused to another identical ordifferent sdAb.

In some embodiments, there is provided a multispecific (such astetraspecific) antigen binding protein comprising: (a) a first antigenbinding portion comprising a first heavy chain variable domain (V_(H))and a first light chain variable domain (V_(L)), wherein the first V_(H)and first V_(L) together form a first antigen-binding site thatspecifically binds a first epitope; (b) a second antigen binding portioncomprising an sdAb that specifically binds a second epitope; (c) a thirdantigen binding portion comprising a third heavy chain variable domain(V_(H)) and a third light chain variable domain (V_(L)), wherein thethird V_(H) and third V_(L) together form a third antigen-binding sitethat specifically binds a third epitope; and (d) a fourth antigenbinding portion comprising a second sdAb that specifically binds afourth epitope; wherein the first antigen binding portion and the secondantigen binding portion are fused to each other, and wherein the thirdantigen binding portion and the fourth antigen binding portion are fusedto each other. An example is shown in FIG. 8 . In some embodiments, oneor more of the sdAbs is each further fused to another identical ordifferent sdAb.

Epitopes and Antigens

Any of the MABPs described herein can specifically bind at least twodifferent epitopes. The at least two different epitopes recognized canbe located on the same antigen, or on different antigens. In someembodiments, the antigens are cell surface molecules. In someembodiments, the antigens are extracellular molecules.

Thus, in some embodiments, there is provided a multispecific (such asbispecific) antigen binding protein comprising: (a) a first antigenbinding portion comprising a heavy chain variable domain (V_(H)) and alight chain variable domain (V_(L)), wherein the V_(H) and V_(L)together form an antigen-binding site that specifically binds a firstantigen, and (b) a second antigen binding portion comprising an sdAbthat specifically binds a second antigen, wherein the first antigenbinding portion and the second antigen binding portion are fused to eachother. In some embodiments, the sdAb is a camelid, humanized, or humansdAb. In some embodiments, the first antigen binding portion comprises aheavy chain comprising the V_(H) and a light chain comprising the V_(L).In some embodiments, the second antigen binding portion is fused to thefirst antigen binding portion at the N-terminus of the heavy chain, theN-terminus of the light chain, the N-terminus of the Fc region, theC-terminus of the heavy chain, or the C-terminus of the light chain. Insome embodiments, the first antigen binding portion comprises afull-length 4-chain antibody. In some embodiments, the second antigenbinding portion is fused to the first antigen binding portionchemically. In some embodiments, the second antigen binding portion isfused to the first antigen binding portion via a peptide bond or apeptide linker. In some embodiments, the peptide linker is no more thanabout 30 (such as no more than about any one of 25, 20, or 15) aminoacids long. In some embodiments, the first antigen binding fragmentcomprises an Fc region, such as an IgG1 Fc or IgG4 Fc.

In some embodiments, the first epitope and/or the second epitope is animmune checkpoint molecule. In some embodiments, the immune checkpointmolecule is a stimulatory immune checkpoint molecule. Exemplarystimulatory immune checkpoint molecules include, but are not limited to,CD28, OX40, ICOS, GITR, 4-1BB, CD27, CD40, CD3, HVEM, and TCR (e.g., MHCclass I or class II molecules). In some embodiments, the immunecheckpoint molecule is an inhibitory immune checkpoint molecule.Exemplary inhibitory immune checkpoint molecules include, but are notlimited to, CTLA-4, TIM-3, A2a Receptor, LAG-3, BTLA, KIR, PD-1, IDO,CD47, and ligands thereof such as B7.1, B7.2, PD-L1, PD-L2, HVEM, B7-H4,NKTR-218, and SIRP-alpha receptor.

Thus, in some embodiments, there is provided a multispecific (such asbispecific) antigen binding protein comprising: (a) a first antigenbinding portion comprising a heavy chain variable domain (V_(H)) and alight chain variable domain (V_(L)), wherein the V_(H) and V_(L)together form an antigen-binding site that specifically binds a firstimmune checkpoint molecule, and (b) a second antigen binding portioncomprising an sdAb that specifically binds a second immune checkpointmolecule, wherein the first antigen binding portion and the secondantigen binding portion are fused to each other. In some embodiments,the sdAb is a camelid, humanized, or human sdAb. In some embodiments,the first immune checkpoint molecule and/or the second immune checkpointmolecule is selected from the group consisting of PD-1, PD-L1, PD-L2,CTLA-4, B7-H3, TIM-3, LAG-3, VISTA, ICOS, 4-1BB, OX40, GITR, and CD40.In some embodiments, the first antigen binding portion comprises a heavychain comprising the V_(H) and a light chain comprising the V_(L). Insome embodiments, the second antigen binding portion is fused to thefirst antigen binding portion at the N-terminus of the heavy chain, theN-terminus of the light chain, the N-terminus of the Fc region, theC-terminus of the heavy chain, or the C-terminus of the light chain. Insome embodiments, the first antigen binding portion comprises afull-length 4-chain antibody. In some embodiments, the second antigenbinding portion is fused to the first antigen binding portionchemically. In some embodiments, the second antigen binding portion isfused to the first antigen binding portion via a peptide bond or apeptide linker. In some embodiments, the peptide linker is no more thanabout 30 (such as no more than about any one of 25, 20, or 15) aminoacids long. In some embodiments, the first antigen binding fragmentcomprises an Fc region, such as an IgG4 Fc.

In some embodiments, the first epitope and/or the second epitope is acell surface antigen. In some embodiments, the cell surface antigen isan antigen on immune effector cells, such as T cells (e.g., helper Tcells, cytotoxic T cells, memory T cells, etc.), B cells, macrophages,and Natural Killer (NK) cells. In some embodiments, the cell surfaceantigen is a T cell surface antigen, such as CD3.

In some embodiments, the cell surface antigen is a tumor antigen. Tumorantigens are proteins that are produced by tumor cells that can elicitan immune response, particularly T-cell mediated immune responses. Theselection of the targeted antigen described herein will depend on theparticular type of cancer to be treated. Exemplary tumor antigensinclude, for example, a glioma-associated antigen, carcinoembryonicantigen (CEA), β-human chorionic gonadotropin, alphafetoprotein (AFP),lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerasereverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, muthsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP,NY-ESO-1, LAGE-la, p53, prostein, PSMA, HER2/neu, survivin andtelomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M,neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF)-I,IGF-II, IGF-I receptor and mesothelin.

In some embodiments, the tumor antigen comprises one or more antigeniccancer epitopes associated with a malignant tumor. Malignant tumorsexpress a number of proteins that can serve as target antigens for animmune attack. These molecules include but are not limited totissue-specific antigens such as MART-1, tyrosinase and gp100 inmelanoma and prostatic acid phosphatase (PAP) and prostate-specificantigen (PSA) in prostate cancer. Other target molecules belong to thegroup of transformation-related molecules such as the oncogeneHER2/Neu/ErbB-2. Yet another group of target antigens are onco-fetalantigens such as carcinoembryonic antigen (CEA). In B-cell lymphoma thetumor-specific idiotype immunoglobulin constitutes a trulytumor-specific immunoglobulin antigen that is unique to the individualtumor. B-cell differentiation antigens such as CD 19, CD20 and CD37 areother candidates for target antigens in B-cell lymphoma.

In some embodiments, the tumor antigen is a tumor-specific antigen (TSA)or a tumor-associated antigen (TAA). A TSA is unique to tumor cells anddoes not occur on other cells in the body. A TAA associated antigen isnot unique to a tumor cell, and instead is also expressed on a normalcell under conditions that fail to induce a state of immunologictolerance to the antigen. The expression of the antigen on the tumor mayoccur under conditions that enable the immune system to respond to theantigen. TAAs may be antigens that are expressed on normal cells duringfetal development, when the immune system is immature, and unable torespond or they may be antigens that are normally present at extremelylow levels on normal cells, but which are expressed at much higherlevels on tumor cells.

Non-limiting examples of TSA or TAA antigens include the following:Differentiation antigens such as MART-1/MelanA (MART-I), gp 100 (Pmel17), tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigenssuch as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5; overexpressedembryonic antigens such as CEA; overexpressed oncogenes and mutatedtumor-suppressor genes such as p53, Ras, HER2/neu; unique tumor antigensresulting from chromosomal translocations; such as BCR-ABL, E2A-PRL,H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barrvirus antigens EBVA and the human papillomavirus (HPV) antigens E6 andE7. Other large, protein-based antigens include TSP-180, MAGE-4, MAGE-5,MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23HI, PSA,TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4,Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG,BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50,CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50,MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS 1, SDCCAG16, TA-90\Mac-2 bindingprotein\cyclophilin C-associated protein, TAAL6, TAG72, TLP, and TPS.

Thus, in some embodiments, there is provided a multispecific (such asbispecific) antigen binding protein comprising: (a) a first antigenbinding portion comprising a heavy chain variable domain (V_(H)) and alight chain variable domain (V_(L)), wherein the V_(H) and V_(L)together form an antigen-binding site that specifically binds a firsttumor antigen, and (b) a second antigen binding portion comprising ansdAb that specifically binds a second tumor antigen, wherein the firstantigen binding portion and the second antigen binding portion are fusedto each other. In some embodiments, the sdAb is a camelid, humanized, orhuman sdAb. In some embodiments, the first tumor antigen and/or thesecond tumor antigen is selected from the group consisting of HER2,BRAF, EGFR, VEGFR2, CD20, RANKL, CD38, and CD52. In some embodiments,the first antigen binding portion comprises a heavy chain comprising theV_(H) and a light chain comprising the V_(L). In some embodiments, thesecond antigen binding portion is fused to the first antigen bindingportion at the N-terminus of the heavy chain, the N-terminus of thelight chain, the N-terminus of the Fc region, the C-terminus of theheavy chain, or the C-terminus of the light chain. In some embodiments,the first antigen binding portion comprises a full-length 4-chainantibody. In some embodiments, the second antigen binding portion isfused to the first antigen binding portion chemically. In someembodiments, the second antigen binding portion is fused to the firstantigen binding portion via a peptide bond or a peptide linker. In someembodiments, the peptide linker is no more than about 30 (such as nomore than about any one of 25, 20, or 15) amino acids long. In someembodiments, the first antigen binding fragment comprises an Fc region,such as an IgG1 Fc.

In some embodiments, there is provided a multispecific (such asbispecific) antigen binding protein comprising: (a) a first antigenbinding portion comprising a heavy chain variable domain (V_(H)) and alight chain variable domain (V_(L)), wherein the V_(H) and V_(L)together form an antigen-binding site that specifically binds a tumorantigen, and (b) a second antigen binding portion comprising an sdAbthat specifically binds a cell surface antigen on an immune effectorcell (such as T cell), wherein the first antigen binding portion and thesecond antigen binding portion are fused to each other. In someembodiments, the sdAb is a camelid, humanized, or human sdAb. In someembodiments, the tumor antigen is selected from the group consisting ofHER2, BRAF, EGFR, VEGFR2, CD20, RANKL, CD38, and CD52. In someembodiments, the first antigen binding portion comprises a heavy chaincomprising the V_(H) and a light chain comprising the V_(L). In someembodiments, the second antigen binding portion is fused to the firstantigen binding portion at the N-terminus of the heavy chain, theN-terminus of the light chain, the N-terminus of the Fc region, theC-terminus of the heavy chain, or the C-terminus of the light chain. Insome embodiments, the first antigen binding portion comprises afull-length 4-chain antibody. In some embodiments, the second antigenbinding portion is fused to the first antigen binding portionchemically. In some embodiments, the second antigen binding portion isfused to the first antigen binding portion via a peptide bond or apeptide linker. In some embodiments, the peptide linker is no more thanabout 30 (such as no more than about any one of 25, 20, or 15) aminoacids long. In some embodiments, the first antigen binding fragmentcomprises an Fc region, such as an IgG1 Fc or IgG4 Fc.

In some embodiments, the first epitope and/or the second epitope is apro-inflammatory molecule. “Pro-inflammatory molecule” refers to anymolecule produced or expressed by an immune cell (such as monocytes,macrophages, lymphocytes and leukocytes) that up-regulates inflammatoryreactions. In some embodiments, the pro-inflammatory molecule is apro-inflammatory cytokine, such as lymphokine, monokine, chemokine, orinterleukin. Exemplary pro-inflammatory molecules include, but are notlimited to, IL-1β, TNF-α, IL-6, IL-6R, IL-5, IL-17, IL-23, IL-22, IL-21,IL-12, and eotaxin-1 (i.e., CCL11).

Thus, in some embodiments, there is provided a multispecific (such asbispecific) antigen binding protein comprising: (a) a first antigenbinding portion comprising a heavy chain variable domain (V_(H)) and alight chain variable domain (V_(L)), wherein the V_(H) and V_(L)together form an antigen-binding site that specifically binds a firstpro-inflammatory molecule, and (b) a second antigen binding portioncomprising an sdAb that specifically binds a second pro-inflammatorymolecule, wherein the first antigen binding portion and the secondantigen binding portion are fused to each other. In some embodiments,the sdAb is a camelid, humanized, or human sdAb. In some embodiments,the first pro-inflammatory molecule and/or the second pro-inflammatorymolecule is selected from the group consisting of IL-1β, TNF-α, IL-5,IL-6, IL-6R, and eotaxin-1. In some embodiments, the first antigenbinding portion comprises a heavy chain comprising the V_(H) and a lightchain comprising the V_(L). In some embodiments, the second antigenbinding portion is fused to the first antigen binding portion at theN-terminus of the heavy chain, the N-terminus of the light chain, theN-terminus of the Fc region, the C-terminus of the heavy chain, or theC-terminus of the light chain. In some embodiments, the first antigenbinding portion comprises a full-length 4-chain antibody. In someembodiments, the second antigen binding portion is fused to the firstantigen binding portion chemically. In some embodiments, the secondantigen binding portion is fused to the first antigen binding portionvia a peptide bond or a peptide linker. In some embodiments, the peptidelinker is no more than about 30 (such as no more than about any one of25, 20, or 15) amino acids long. In some embodiments, the first antigenbinding fragment comprises an Fc region, such as an IgG1 Fc.

In some embodiments, the first epitope and/or the second epitope is anangiogenic factor, such as Ang2 and VEGF. Thus, in some embodiments,there is provided a multispecific (such as bispecific) antigen bindingprotein comprising: (a) a first antigen binding portion comprising aheavy chain variable domain (V_(H)) and a light chain variable domain(V_(L)), wherein the V_(H) and V_(L) together form an antigen-bindingsite that specifically binds a first angiogenic factor, and (b) a secondantigen binding portion comprising an sdAb that specifically binds asecond angiogenic factor, wherein the first antigen binding portion andthe second antigen binding portion are fused to each other.

Fusion Polypeptides

The first antigen binding portion and the second antigen binding portionof the MABP are fused (i.e., covalently linked) to each other. Thus, theMABPs of the present application comprise one or more fusionpolypeptides. Each fusion polypeptide may comprise the second antigenbinding portion and a polypeptide from the first antigen bindingportion.

The first antigen binding portion and the second antigen binding portionmay be linked directly by a single chemical bond (such as peptide bond)or via a peptide linker. The second antigen binding portion may be fusedat either the N-terminus or the C-terminus of any one (including each)polypeptide of the first antigen binding portion, or may be fused at aninternal position of any one (including each) polypeptide of the firstantigen binding portion, such as at the N-terminus of the Fc region inthe heavy chain of the first antigen binding portion. The fusionpolypeptides may be obtained either recombinantly or chemically. In someembodiments, the C-terminus of the second antigen binding portion isfused to the N-terminus of any (including each) polypeptide of the firstantigen binding portion via a chemical bond (such as peptide bond) or apeptide linker. In some embodiments, the N-terminus of the secondantigen binding portion is fused to the C-terminus of any (includingeach) polypeptide of the first antigen binding portion via a chemicalbond (such as peptide bond) or a peptide linker. In some embodiments,the second antigen binding portion is fused to the first antigen bindingportion via a chemical bond that is not a peptide bond involving themain chain chemical groups of amino acids.

In some embodiments, the first antigen binding portion comprises asingle-chain antibody fragment comprising the V_(H) and V_(L). In someembodiments, the first antigen binding portion comprises an scFv. Insome embodiments, the MABP comprises a fusion polypeptide comprising inthe N-terminus to C-terminus direction: the second antigen bindingportion comprising the sdAb, an optional peptide linker, the V_(H)domain and the V_(L) domain. In some embodiments, the MABP comprises afusion polypeptide comprising in the N-terminus to C-terminus direction:the second antigen binding portion comprising the sdAb, an optionalpeptide linker, the V_(L) domain and the V_(H) domain. In someembodiments, the MABP comprises a fusion polypeptide comprising in theN-terminus to C-terminus direction: the V_(H) domain, the V_(L) domain,an optional peptide linker, and the second antigen binding portioncomprising the sdAb. In some embodiments, the MABP comprises a fusionpolypeptide comprising in the N-terminus to C-terminus direction: theV_(L) domain, the V_(H) domain, an optional peptide linker, and thesecond antigen binding portion comprising the sdAb.

In some embodiments, the first antigen binding portion comprises a heavychain comprising the V_(H) domain, and a light chain comprising theV_(L) domain. In some embodiments, the heavy chain further comprises oneor more heavy chain constant domains, such as C_(H)1, C_(H)2, C_(H)4,and C_(H)3, and/or an antibody hinge region (HR). In some embodiments,the light chain further comprises a light chain constant domain (C_(L)),such as the lambda C_(L) domain or kappa C_(L) domain. In someembodiments, the N-terminus of the second antigen binding portion isfused to the C-terminus of the heavy chain. In some embodiments, theC-terminus of the second antigen binding portion is fused to theN-terminus of the heavy chain. In some embodiments, the N-terminus ofthe second antigen binding portion is fused to the C-terminus of thelight chain. In some embodiments, the C-terminus of the second antigenbinding portion is fused to the N-terminus of the light chain. In someembodiments, the MABP comprises a first polypeptide comprising from theN-terminus to the C-terminus: the heavy chain, an optional peptidelinker, and the second antigen binding portion comprising the sdAb; anda second polypeptide comprising the light chain. In some embodiments,the MABP comprises a first polypeptide comprising from the N-terminus tothe C-terminus: the second antigen binding portion comprising the sdAb,an optional peptide linker, and the heavy chain; and a secondpolypeptide comprising the light chain. In some embodiments, the MABPcomprises a first polypeptide comprising from the N-terminus to theC-terminus: the light chain, an optional peptide linker, and the secondantigen binding portion comprising the sdAb; and a second polypeptidecomprising the heavy chain. In some embodiments, the MABP comprises afirst polypeptide comprising from the N-terminus to the C-terminus: thesecond antigen binding portion comprising the sdAb, an optional peptidelinker, and the light chain; and a second polypeptide comprising theheavy chain.

In some embodiments, the first antigen binding portion comprises afull-length antibody consisting of two heavy chains and two lightchains. In some embodiments, the full-length antibody is a full-lengthmonoclonal antibody consisting of two identical heavy chains and twoidentical light chains. In some embodiments, the MABP comprises twoidentical first polypeptides each comprising from the N-terminus to theC-terminus: the heavy chain, an optional peptide linker, and the secondantigen binding portion comprising the sdAb; and two second polypeptideseach comprising the light chain (see, for example, FIG. 4 ). In someembodiments, the MABP comprises two identify first polypeptides eachcomprising from the N-terminus to the C-terminus: the second antigenbinding portion comprising the sdAb, an optional peptide linker, and theheavy chain; and two identical second polypeptides each comprising thelight chain (see, for example, FIG. 9 ). In some embodiments, the MABPcomprises two identical first polypeptides each comprising from theN-terminus to the C-terminus: the light chain, an optional peptidelinker, and the second antigen binding portion comprising the sdAb; andtwo identical second polypeptides each comprising the heavy chain (see,for example, FIG. 11 ). In some embodiments, the MABP comprises twoidentical first polypeptides each comprising from the N-terminus to theC-terminus: the second antigen binding portion comprising the sdAb, anoptional peptide linker, and the light chain; and two identical secondpolypeptides comprising the heavy chain (see, for example, FIG. 13 ).

In some embodiments, the MABP comprises: (a) a full-length antibodyconsisting of two heavy chains and two light chains, wherein thefull-length antibody specifically recognizes a first epitope; (b) afirst sdAb that specifically recognizes a second epitope; and (c) asecond sdAb that specifically recognizes a third epitope, wherein theC-terminus of the first sdAb is fused to the N-terminus of each heavychain, and wherein the N-terminus of the second sdAb is fused to theC-terminus of each heavy chain. In some embodiments, the MABP comprisestwo identical first polypeptides each comprising from the N-terminus tothe C-terminus: the first sdAb, an optional peptide linker, the heavychain, an optional peptide linker, and the second sdAb; and twoidentical second polypeptides each comprising the light chain. See, forexample, FIG. 15 .

In some embodiments, the MABP comprises: (a) a full-length antibodyconsisting of two heavy chains and two light chains, wherein thefull-length antibody specifically recognizes a first epitope; (b) afirst sdAb that specifically recognizes a second epitope; and (c) asecond sdAb that specifically recognizes a third epitope, wherein theC-terminus of the first sdAb is fused to the N-terminus of each lightchain, and wherein the N-terminus of the second sdAb is fused to theC-terminus of each heavy chain. In some embodiments, the MABP comprisestwo identical first polypeptides each comprising from the N-terminus tothe C-terminus: the heavy chain, an optional peptide linker, and thesecond sdAb; and two identical second polypeptides each comprising thefirst sdAb, an optional peptide linker, and the light chain. See, forexample, FIG. 16 .

In some embodiments, the MABP comprises: (a) a full-length antibodyconsisting of a first and a second heavy chains and a first and a secondlight chains, wherein the full-length antibody specifically recognizes afirst epitope; (b) a first sdAb that specifically recognizes a secondepitope; (c) a second sdAb that specifically recognizes a third epitope;(d) a third sdAb that specifically recognizes a fourth epitope; and (e)a fourth sdAb that specifically recognizes a fifth epitope; wherein theC-terminus of the first sdAb is fused to the N-terminus of the firstlight chain, wherein the C-terminus of the second sdAb is fused to theN-terminus of the second light chain, wherein the C-terminus of thethird sdAb is fused to the N-terminus of the first heavy chain, andwherein the C-terminus of the fourth sdAb is fused to the N-terminus ofthe second heavy chain. In some embodiments, the MABP comprises twoidentical first polypeptides each comprising from the N-terminus to theC-terminus: the third or the fourth sdAb, an optional peptide linker,and the heavy chain; and two identical second polypeptides eachcomprising the first or the second sdAb, an optional peptide linker, andthe light chain. See, for example, FIG. 17 .

In some embodiments, the MABP comprises: (a) a full-length antibodyconsisting of two heavy chains and two light chains, wherein thefull-length antibody specifically recognizes a first epitope; (b) afirst sdAb that specifically recognizes a second epitope; (c) a secondsdAb that specifically recognizes a third epitope; (d) a third sdAb thatspecifically recognizes a fourth epitope; and (e) a fourth sdAb thatspecifically recognizes a fifth epitope; wherein the C-terminus of thefirst sdAb is fused to the N-terminus of the second sdAb, and theC-terminus of the second sdAb is fused to the N-terminus of one heavychain, and wherein the C-terminus of the third sdAb is fused to theN-terminus of the fourth sdAb, and the C-terminus of the fourth sdAb isfused to the N-terminus of the other heavy chain. In some embodiments,the MABP comprises two identical first polypeptides each comprising fromthe N-terminus to the C-terminus: the first or the third sdAb, anoptional peptide linker, the second or the fourth sdAb, an optionalpeptide linker, and the heavy chain; and two identical secondpolypeptides each comprising the light chain. See, for example, FIG. 18.

In some embodiments, the MABP comprises: (a) a full-length antibodyconsisting of two heavy chains and two light chains, wherein thefull-length antibody specifically recognizes a first epitope; (b) afirst sdAb that specifically recognizes a second epitope; and (c) asecond sdAb that specifically recognizes a third epitope, wherein theN-terminus of the first or the second sdAb is fused to the C-terminus ofthe C_(H)1 region of the heavy chain, and the C-terminus of the first orthe second sdAb is fused to the N-terminus of the C_(H)2 region of theheavy chain. In some embodiments, the MABP comprises two identical firstpolypeptides each comprising from the N-terminus to the C-terminus:V_(H)—C_(H)1-an optional peptide linker-sdAb-C_(H)2-C_(H)3; and twoidentical second polypeptides each comprising the light chain. See, forexample, FIG. 19 .

In some embodiments, the MABP comprises: (a) a first scFv thatspecifically recognizes a first epitope; (b) a second scFv thatspecifically recognizes a second epitope; (c) an Fc region; (d) a firstsdAb that specifically recognizes a third epitope; and (d) a second sdAbthat specifically recognizes a fourth epitope, wherein the N-terminus ofeach sdAb is fused to the C-terminus of an scFv and the C-terminus ofthe sdAb is fused to the N-terminus of the Fc region. In someembodiments, the MABP comprises two identical polypeptides eachcomprising from the N-terminus to the C-terminus: scFv-an optionalpeptide linker-sdAb-CH2-CH3. See, for example, FIG. 20 .

In some embodiments, the MABP comprises: (a) a first Fab thatspecifically recognizes a first epitope; (b) a second Fab thatspecifically recognizes a second epitope; (c) an Fc region; (d) a firstFab-like domain comprising a first sdAb that specifically recognizes athird epitope and a second sdAb that specifically recognizes a fourthepitope; (e) a second Fab-like domain comprising a third sdAb thatspecifically recognizes a fifth epitope and a fourth sdAb thatspecifically recognizes a sixth epitope, wherein the N-termini of eachFab-like domain are fused to the C-termini of a Fab and one of the twoC-termini of the Fab-like domain is fused to the N-terminus of the Fcregion. In some embodiments, the MABP comprises two identical firstpolypeptides each comprising from the N-terminus to the C-terminus:V_(H)-C_(H)1-an optional peptide linker-sdAb-C_(H)1-C_(H)2-C_(H)3; andtwo identical second polypeptides each comprising from the N-terminus tothe C-terminus: V_(L)-C_(L)-an optional peptide linker-sdAb-C_(L). See,for example, FIG. 21 .

In some embodiments, the MABP comprises: (a) a first scFv thatspecifically recognizes a first epitope; (b) a second scFv thatspecifically recognizes a second epitope; (c) an Fc region; (d) a firstFab-like domain comprising a first sdAb that specifically recognizes athird epitope and a second sdAb that specifically recognizes a fourthepitope; (e) a second Fab-like domain comprising a third sdAb thatspecifically recognizes a fifth epitope and a fourth sdAb thatspecifically recognizes a sixth epitope, wherein one of the twoN-termini of each Fab-like domain is fused to the C-terminus of an scFvand one of the two C-termini of the sdAb is fused to an N-terminus ofthe Fc region. In some embodiments, the MABP comprises two identicalfirst polypeptides each comprising from the N-terminus to theC-terminus: scFv-an optional peptide linker-sdAb-C_(H)1-C_(H)2-C_(H)3;and two identical second polypeptides each comprising from theN-terminus to the C-terminus: sdAb-C_(L). See, for example, FIG. 22 .

Thus, in some embodiments, there is provided a multispecific (such asbispecific) antigen binding protein comprising: (a) a first antigenbinding portion comprising a heavy chain comprising a heavy chainvariable domain (V_(H)) and a light chain comprising a light chainvariable domain (V_(L)), wherein the V_(H) and V_(L) together form anantigen-binding site that specifically binds a first epitope, and (b) asecond antigen binding portion comprising an sdAb that specificallybinds a second epitope, wherein the N-terminus of the second antigenbinding portion is fused to the C-terminus of the heavy chain of thefirst antigen binding portion via a peptide bond or a peptide linker. Insome embodiments, the sdAb is a camelid, humanized, or human sdAb. Insome embodiments, the first epitope is from a first immune checkpointmolecule, and the second epitope is from a second immune checkpointmolecule. In some embodiments, the first epitope is from a first tumorantigen, and the second epitope is from a second tumor antigen. In someembodiments, the first epitope is from a tumor antigen, and the secondepitope is from a cell surface molecule, such as CD3. In someembodiments, the first epitope is from a first pro-inflammatorymolecule, and the second epitope is from a second pro-inflammatorymolecule. In some embodiments, the first epitope is from a firstangiogenic factor, and the second epitope is from a second angiogenicfactor. In some embodiments, the peptide linker is no more than about 30(such as no more than about any one of 25, 20, or 15) amino acids long.In some embodiments, the first antigen binding fragment comprises an Fcregion, such as an IgG1 Fc or IgG4 Fc.

In some embodiments, there is provided a multispecific (such asbispecific) antigen binding protein comprising: (a) a first antigenbinding portion comprising a heavy chain comprising a heavy chainvariable domain (V_(H)) and a light chain comprising a light chainvariable domain (V_(L)), wherein the V_(H) and V_(L) together form anantigen-binding site that specifically binds a first epitope, and (b) asecond antigen binding portion comprising an sdAb that specificallybinds a second epitope, wherein the C-terminus of the second antigenbinding portion is fused to the N-terminus of the heavy chain of thefirst antigen binding portion via a peptide bond or a peptide linker. Insome embodiments, the sdAb is a camelid, humanized, or human sdAb. Insome embodiments, the first epitope is from a first immune checkpointmolecule, and the second epitope is from a second immune checkpointmolecule. In some embodiments, the first epitope is from a first tumorantigen, and the second epitope is from a second tumor antigen. In someembodiments, the first epitope is from a tumor antigen, and the secondepitope is from a cell surface molecule, such as CD3. In someembodiments, the first epitope is from a first pro-inflammatorymolecule, and the second epitope is from a second pro-inflammatorymolecule. In some embodiments, the first epitope is from a firstangiogenic factor, and the second epitope is from a second angiogenicfactor. In some embodiments, the peptide linker is no more than about 30(such as no more than about any one of 25, 20, or 15) amino acids long.In some embodiments, the first antigen binding fragment comprises an Fcregion, such as an IgG1 Fc or IgG4 Fc.

In some embodiments, there is provided a multispecific (such asbispecific) antigen binding protein comprising: (a) a first antigenbinding portion comprising a heavy chain comprising a heavy chainvariable domain (V_(H)) and a light chain comprising a light chainvariable domain (V_(L)), wherein the V_(H) and V_(L) together form anantigen-binding site that specifically binds a first epitope, and (b) asecond antigen binding portion comprising an sdAb that specificallybinds a second epitope, wherein the N-terminus of the second antigenbinding portion is fused to the C-terminus of the light chain of thefirst antigen binding portion via a peptide bond or a peptide linker. Insome embodiments, the sdAb is a camelid, humanized, or human sdAb. Insome embodiments, the first epitope is from a first immune checkpointmolecule, and the second epitope is from a second immune checkpointmolecule. In some embodiments, the first epitope is from a first tumorantigen, and the second epitope is from a second tumor antigen. In someembodiments, the first epitope is from a tumor antigen, and the secondepitope is from a cell surface molecule, such as CD3. In someembodiments, the first epitope is from a first pro-inflammatorymolecule, and the second epitope is from a second pro-inflammatorymolecule. In some embodiments, the first epitope is from a firstangiogenic factor, and the second epitope is from a second angiogenicfactor. In some embodiments, the peptide linker is no more than about 30(such as no more than about any one of 25, 20, or 15) amino acids long.In some embodiments, the first antigen binding fragment comprises an Fcregion, such as an IgG1 Fc or IgG4 Fc.

In some embodiments, there is provided a multispecific (such asbispecific) antigen binding protein comprising: (a) a first antigenbinding portion comprising a heavy chain comprising a heavy chainvariable domain (V_(H)) and a light chain comprising a light chainvariable domain (V_(L)), wherein the V_(H) and V_(L) together form anantigen-binding site that specifically binds a first epitope, and (b) asecond antigen binding portion comprising an sdAb that specificallybinds a second epitope, wherein the C-terminus of the second antigenbinding portion is fused to the N-terminus of the light chain of thefirst antigen binding portion via a peptide bond or a peptide linker. Insome embodiments, the sdAb is a camelid, humanized, or human sdAb. Insome embodiments, the first epitope is from a first immune checkpointmolecule, and the second epitope is from a second immune checkpointmolecule. In some embodiments, the first epitope is from a first tumorantigen, and the second epitope is from a second tumor antigen. In someembodiments, the first epitope is from a tumor antigen, and the secondepitope is from a cell surface molecule, such as CD3. In someembodiments, the first epitope is from a first pro-inflammatorymolecule, and the second epitope is from a second pro-inflammatorymolecule. In some embodiments, the first epitope is from a firstangiogenic factor, and the second epitope is from a second angiogenicfactor. In some embodiments, the peptide linker is no more than about 30(such as no more than about any one of 25, 20, or 15) amino acids long.In some embodiments, the first antigen binding fragment comprises an Fcregion, such as an IgG1 Fc or IgG4 Fc.

In some embodiments, there is provided a multispecific (such asbispecific) antigen binding protein comprising: (a) a first antigenbinding portion comprising a full-length antibody comprising two heavychains and two light chains, wherein the full-length antibodyspecifically binds a first epitope, and (b) a second antigen bindingportion comprising an sdAb that specifically binds a second epitope,wherein the N-terminus of the second antigen binding portion is fused tothe C-terminus of one or each of the two heavy chains of the firstantigen binding portion via a peptide bond or a peptide linker. In someembodiments, the sdAb is a camelid, humanized, or human sdAb. In someembodiments, the first epitope is from a first immune checkpointmolecule, and the second epitope is from a second immune checkpointmolecule. In some embodiments, the first epitope is from a first tumorantigen, and the second epitope is from a second tumor antigen. In someembodiments, the first epitope is from a tumor antigen, and the secondepitope is from a cell surface molecule, such as CD3. In someembodiments, the first epitope is from a first pro-inflammatorymolecule, and the second epitope is from a second pro-inflammatorymolecule. In some embodiments, the first epitope is from a firstangiogenic factor, and the second epitope is from a second angiogenicfactor. In some embodiments, the peptide linker is no more than about 30(such as no more than about any one of 25, 20, or 15) amino acids long.In some embodiments, the first antigen binding fragment comprises an Fcregion, such as an IgG1 Fc or IgG4 Fc.

In some embodiments, there is provided a multispecific (such asbispecific) antigen binding protein comprising: (a) a first antigenbinding portion comprising a full-length antibody comprising two heavychains and two light chains, wherein the full-length antibodyspecifically binds a first epitope, and (b) a second antigen bindingportion comprising an sdAb that specifically binds a second epitope,wherein the C-terminus of the second antigen binding portion is fused tothe N-terminus of one or each of the two heavy chains of the firstantigen binding portion via a peptide bond or a peptide linker. In someembodiments, the sdAb is a camelid, humanized, or human sdAb. In someembodiments, the first epitope is from a first immune checkpointmolecule, and the second epitope is from a second immune checkpointmolecule. In some embodiments, the first epitope is from a first tumorantigen, and the second epitope is from a second tumor antigen. In someembodiments, the first epitope is from a tumor antigen, and the secondepitope is from a cell surface molecule, such as CD3. In someembodiments, the first epitope is from a first pro-inflammatorymolecule, and the second epitope is from a second pro-inflammatorymolecule. In some embodiments, the first epitope is from a firstangiogenic factor, and the second epitope is from a second angiogenicfactor. In some embodiments, the peptide linker is no more than about 30(such as no more than about any one of 25, 20, or 15) amino acids long.In some embodiments, the first antigen binding fragment comprises an Fcregion, such as an IgG1 Fc or IgG4 Fc.

In some embodiments, there is provided a multispecific (such asbispecific) antigen binding protein comprising: (a) a first antigenbinding portion comprising a full-length antibody comprising two heavychains and two light chains, wherein the full-length antibodyspecifically binds a first epitope, and (b) a second antigen bindingportion comprising an sdAb that specifically binds a second epitope,wherein the N-terminus of the second antigen binding portion is fused tothe C-terminus of one or each of the two light chains of the firstantigen binding portion via a peptide bond or a peptide linker. In someembodiments, the sdAb is a camelid, humanized, or human sdAb. In someembodiments, the first epitope is from a first immune checkpointmolecule, and the second epitope is from a second immune checkpointmolecule. In some embodiments, the first epitope is from a first tumorantigen, and the second epitope is from a second tumor antigen. In someembodiments, the first epitope is from a tumor antigen, and the secondepitope is from a cell surface molecule, such as CD3. In someembodiments, the first epitope is from a first pro-inflammatorymolecule, and the second epitope is from a second pro-inflammatorymolecule. In some embodiments, the first epitope is from a firstangiogenic factor, and the second epitope is from a second angiogenicfactor. In some embodiments, the peptide linker is no more than about 30(such as no more than about any one of 25, 20, or 15) amino acids long.In some embodiments, the first antigen binding fragment comprises an Fcregion, such as an IgG1 Fc or IgG4 Fc.

In some embodiments, there is provided a multispecific (such asbispecific) antigen binding protein comprising: (a) a first antigenbinding portion comprising a full-length antibody comprising two heavychains and two light chains, wherein the full-length antibodyspecifically binds a first epitope, and (b) a second antigen bindingportion comprising an sdAb that specifically binds a second epitope,wherein the C-terminus of the second antigen binding portion is fused tothe N-terminus of one or each of the two light chains of the firstantigen binding portion via a peptide bond or a peptide linker. In someembodiments, the first epitope is from a first immune checkpointmolecule, and the second epitope is from a second immune checkpointmolecule. In some embodiments, the sdAb is a camelid, humanized, orhuman sdAb. In some embodiments, the first epitope is from a first tumorantigen, and the second epitope is from a second tumor antigen. In someembodiments, the first epitope is from a tumor antigen, and the secondepitope is from a cell surface molecule, such as CD3. In someembodiments, the first epitope is from a first pro-inflammatorymolecule, and the second epitope is from a second pro-inflammatorymolecule. In some embodiments, the first epitope is from a firstangiogenic factor, and the second epitope is from a second angiogenicfactor. In some embodiments, the peptide linker is no more than about 30(such as no more than about any one of 25, 20, or 15) amino acids long.In some embodiments, the first antigen binding fragment comprises an Fcregion, such as an IgG1 Fc or IgG4 Fc.

The MABPs described herein may comprise one or more peptide linkerssituated between the first antigen binding portion and the secondantigen binding portion. In some embodiments, the peptide linker betweenthe heavy chain polypeptide of the first antigen binding portion and thesecond antigen binding portion is the same as the peptide linker betweenthe light chain polypeptide of the first antigen binding portion and thesecond antigen binding portion. In some embodiments, the peptide linkerbetween the heavy chain polypeptide of the first antigen binding portionand the second antigen binding portion is different from the peptidelinker between the light chain polypeptide of the first antigen bindingportion and the second antigen binding portion. In some embodiments, thefirst antigen binding portion and the second antigen binding portion aredirectly fused to each other without a peptide linker disposedtherebetween.

The various antigen binding portions of the MABPs may be fused to eachother via a peptide linker. The peptide linkers connecting differentantigen binding portions may be the same or different. Each peptidelinker can be optimized individually. The peptide linker can be of anysuitable length. In some embodiments, the peptide linker is at leastabout any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 25, 30, 35, 40, 50 or more amino acids long. In someembodiments, the peptide linker is no more than about any of 50, 40, 35,30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 orfewer amino acids long. In some embodiments, the length of the peptidelinker is any of about 1 amino acid to about 10 amino acids, about 1amino acids to about 20 amino acids, about 1 amino acid to about 30amino acids, about 5 amino acids to about 15 amino acids, about 10 aminoacids to about 25 amino acids, about 5 amino acids to about 30 aminoacids, about 10 amino acids to about 30 amino acids long, about 30 aminoacids to about 50 amino acids, or about 1 amino acid to about 50 aminoacids.

The peptide linker may have a naturally occurring sequence, or anon-naturally occurring sequence. For example, a sequence derived fromthe hinge region of heavy chain only antibodies may be used as thelinker. See, for example, WO1996/34103. In some embodiments, the peptidelinker is a flexible linker. Exemplary flexible linkers include glycinepolymers (G)_(n), glycine-serine polymers (including, for example,(GS)_(n) (SEQ ID NO: 9), (GSGGS)_(n) (SEQ ID NO: 10) and (GGGS)_(n) (SEQID NO: 11), where n is an integer of at least one), glycine-alaninepolymers, alanine-serine polymers, and other flexible linkers known inthe art. In some embodiments, the peptide linker comprises the aminoacid sequence GGGGSGGGS (SEQ ID NO: 1). In some embodiments, the peptidelinker comprises the hinge region of an IgG, such as the hinge region ofhuman IgG1. In some embodiments, the peptide linker comprises the aminoacid sequence EPKSCDKTHTCPPCP (SEQ ID NO: 8). In some embodiments, thepeptide linker comprises a modified sequence derived from the hingeregion of an IgG, such as the hinge region of human IgG1. For example,one or more cysteines in the hinge region of an IgG may be replaced witha serine. In some embodiments, the peptide linker comprises the aminoacid sequence EPKSSDKTHTSPPSP (SEQ ID NO: 12).

In some embodiments, the first antigen binding portion and the secondantigen binding portion are fused to each other chemically. For example,the second antigen binding portion and one or more polypeptides of thefirst antigen binding portion may be conjugated using one or morereactive sites via a linking group. Reactive sites in polypeptides thatare useful for chemical conjugation are well known in the art,including, but not limited to primary amino groups present on amino acidresidue such as the epsilon amino group of lysine, and the alpha aminogroup of N-terminal amino acids, thiol groups in cysteine residues, thecarboxylic group of the C-terminal amino acids, and carbohydrate groupsin glycosylated antibodies. In some embodiments, the reactive site isintroduced into the second antigen binding portion or the first antigenbinding portion by site-directed mutagenesis, incorporation ofselenocysteines or unnatural amino acids, incorporation of bifunctionallinkers (such as bis-alkylating reagents), and/or glycoengineering. Insome embodiments, one or more primary amino groups of a polypeptide canbe converted to a thiol-containing group (e.g., from a cysteine orhomocysteine residue), an electrophilic unsaturated group such as amaleimide group, or halogenated group such as a bromoacetyl group, forconjugation to thiol reactive polypeptides. Any linking groups andconjugation methods known in the art can be used to chemically fuse thesecond antigen binding portion to the first antigen binding portion. Insome embodiments, the conjugation can be achieved, for example, by usingsuccinimide esters (such as succinimidyl4-[N-maleimidomethyl]cyclohexane carboxylate (SMCC), orN-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS)), glutaraldehyde,carbodiimide (such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide(EDCI)), benzidine (BDB), periodate, or isothiocyanate (such as N-acetylhomocysteine thiolactone (NAHT)).

Antigen Binding Portion Comprising Single-Domain Antibody

The MABPs of the present application comprise at least one antigenbinding portion comprising an sdAb. Exemplary sdAbs include, but are notlimited to, heavy chain variable domains from heavy-chain onlyantibodies (e.g., V_(H)H or V_(NAR)), binding molecules naturally devoidof light chains, single domains (such as V_(H) or V_(L)) derived fromconventional 4-chain antibodies, humanized heavy-chain only antibodies,human sdAbs produced by transgenic mice or rats expressing human heavychain segments, and engineered domains and single domain scaffolds otherthan those derived from antibodies. Any sdAbs known in the art ordeveloped by the inventors may be used to construct the MABPs of thepresent application. The sdAbs may be derived from any speciesincluding, but not limited to mouse, rat, human, camel, llama, lamprey,fish, shark, goat, rabbit, and bovine. Single-domain antibodiescontemplated herein also include naturally occurring sdAb molecules fromspecies other than Camelidae and sharks.

In some embodiments, the sdAb is derived from a naturally occurringsingle-domain antigen binding molecule known as heavy chain antibodydevoid of light chains (also referred herein as “heavy chain onlyantibodies”). Such single domain molecules are disclosed in WO 94/04678and Hamers-Casterman, C. et al. (1993) Nature 363:446-448, for example.For clarity reasons, the variable domain derived from a heavy chainmolecule naturally devoid of light chain is known herein as a V_(H)H todistinguish it from the conventional VH of four chain immunoglobulins.Such a V_(H)H molecule can be derived from antibodies raised inCamelidae species, for example, camel, llama, vicuna, dromedary, alpacaand guanaco. Other species besides Camelidae may produce heavy chainmolecules naturally devoid of light chain, and such V_(H)Hs are withinthe scope of the present application.

V_(H)H molecules from Camelids are about 10 times smaller than IgGmolecules. They are single polypeptides and can be very stable,resisting extreme pH and temperature conditions. Moreover, they can beresistant to the action of proteases which is not the case forconventional antibodies. Furthermore, in vitro expression of V_(H)H sproduces high yield, properly folded functional V_(H)Hs. In addition,antibodies generated in Camelids can recognize epitopes other than thoserecognized by antibodies generated in vitro through the use of antibodylibraries or via immunization of mammals other than Camelids (see, forexample, WO9749805). As such, MABPs comprising one or more V_(H)Hdomains may interact more efficiently with targets than conventionalantibodies. Since V_(H)Hs are known to bind into ‘unusual’ epitopes suchas cavities or grooves, the affinity of MABPs comprising such V_(H)Hsmay be more suitable for therapeutic treatment than conventionalmultispecific polypeptides.

In some embodiments, there is provided a multispecific (such asbispecific) antigen binding protein comprising: (a) a first antigenbinding portion comprising a heavy chain variable domain (V_(H)) and alight chain variable domain (V_(L)), wherein the V_(H) and V_(L)together form an antigen-binding site that specifically binds a firstepitope, and (b) a second antigen binding portion comprising a V_(H)Hdomain that specifically binds a second epitope, wherein the firstantigen binding portion and the second antigen binding portion are fusedto each other. In some embodiments, the first epitope is from a firstimmune checkpoint molecule, and the second epitope is from a secondimmune checkpoint molecule. In some embodiments, the first epitope isfrom a first tumor antigen, and the second epitope is from a secondtumor antigen. In some embodiments, the first epitope is from a tumorantigen, and the second epitope is from a cell surface molecule, such asCD3. In some embodiments, the first epitope is from a firstpro-inflammatory molecule, and the second epitope is from a secondpro-inflammatory molecule. In some embodiments, the first antigenbinding portion comprises a heavy chain comprising the V_(H) and a lightchain comprising the V_(L). In some embodiments, the V_(H)H domain ishumanized. In some embodiments, the second antigen binding portion isfused to the first antigen binding portion at the N-terminus of theheavy chain, the N-terminus of the light chain, the N-terminus of the Fcregion, the C-terminus of the heavy chain, or the C-terminus of thelight chain. In some embodiments, the first antigen binding portioncomprises a full-length 4-chain antibody. In some embodiments, thesecond antigen binding portion is fused to the first antigen bindingportion chemically. In some embodiments, the second antigen bindingportion is fused to the first antigen binding portion via a peptide bondor a peptide linker. In some embodiments, the peptide linker is no morethan about 30 (such as no more than about any one of 25, 20, or 15)amino acids long. In some embodiments, the first antigen bindingfragment comprises an Fc region, such as an IgG1 Fc or IgG4 Fc.

In some embodiments, the sdAb is derived from a variable region of theimmunoglobulin found in cartilaginous fish. For example, the sdAb can bederived from the immunoglobulin isotype known as Novel Antigen Receptor(NAR) found in the serum of shark. Methods of producing single domainmolecules derived from a variable region of NAR (“IgNARs”) are describedin WO 03/014161 and Streltsov (2005) Protein Sci. 14:2901-2909.

In some embodiments, the sdAb is recombinant, CDR-grafted, humanized,camelized, de-immunized and/or in vitro generated (e.g., selected byphage display). In some embodiments, the sdAb is a human sdAb producedby transgenic mice or rats expressing human heavy chain segments. See,e.g. US20090307787A1, U.S. Pat. No. 8,754,287, US20150289489A1,US20100122358A1, and WO2004049794. In some embodiments, the sdAb isaffinity matured.

SdAbs comprising a V_(H)H domain can be humanized to have human-likesequences. In some embodiments, the FR regions of the V_(H)H domain usedherein comprise at least about any one of 50%, 60%, 70%, 80%, 90%, 95%or more of amino acid sequence homology to human VH framework regions.One exemplary class of humanized V_(H)H domains is characterized in thatthe V_(H)Hs carry an amino acid from the group consisting of glycine,alanine, valine, leucine, isoleucine, proline, phenylalanine, tyrosine,tryptophan, methionine, serine, threonine, asparagine, or glutamine atposition 45, such as, for example, L45 and a tryptophan at position 103,according to the Kabat numbering. As such, polypeptides belonging tothis class show a high amino acid sequence homology to human VHframework regions and said polypeptides might be administered to a humandirectly without expectation of an unwanted immune response therefrom,and without the burden of further humanization.

Another exemplary class of humanized Camelidae sdAbs has been describedin WO 03/035694 and contains hydrophobic FR2 residues typically found inconventional antibodies of human origin or from other species, butcompensating this loss in hydrophilicity by the charged arginine residueon position 103 that substitutes the conserved tryptophan residuepresent in V_(H) from double-chain antibodies. As such, peptidesbelonging to these two classes show a high amino acid sequence homologyto human V_(H) framework regions and said peptides might be administeredto a human directly without expectation of an unwanted immune responsetherefrom, and without the burden of further humanization.

In some embodiments, the MABP comprises a naturally produced sdAb or aderivative thereof, such as a Camelid sdAb, or a humanized sdAb derivedfrom a Camelid sdAb. In some embodiments, the sdAb is obtained fromllama. In some embodiments, the sdAb is further engineered to removesequences not normally found in human antibodies (such as CDR regions orCDR-FR junctions).

In some embodiments, the MABP comprises a Fab-like domain comprising afirst polypeptide chain comprising a first sdAb (such as V_(H)H) fusedto a C_(H)1 domain, and a second polypeptide chain comprising a secondsdAb (such as V_(H)H) fused to a C_(L) domain. In some embodiments, thefirst sdAb and the second sdAb specifically bind to the same epitope. Insome embodiments, the first sdAb and the second sdAb specifically bindto different epitopes. In some embodiments, each polypeptide chain ofthe Fab-like domain is fused to the N-terminus, C-terminus or aninternal position of a polypeptide chain of the first antigen bindingportion. In some embodiments, one of the two polypeptide chain of theFab-like domain is fused to the N-terminus, C-terminus or an internalposition of a polypeptide chain of the first antigen binding portion. Insome embodiments, the MABP comprises two or more Fab-like domains.

In some embodiments, the MABP comprises an antigen binding portioncomprising an sdAb having a suitable affinity to its epitope. Forexample, the affinity of the sdAb may affect the overall affinity andavidity of the MABP to the target cell or tissue, which may furtheraffect the efficacy of the MABP. In some embodiments, the sdAb binds itsepitope with high affinity. A high-affinity sdAb binds its epitope witha dissociation constant (Kd) in the low nanomolar (10⁻⁹ M) range, suchas no more than about any of 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM, 0.2nM, 0.1 nM, 0.05 nM, 0.02 nM, 0.01 nM, 5 pM, 2 pM, 1 pM or less. In someembodiments, the sdAb binds its epitope with low affinity. Alow-affinity sdAb binds its epitope with a Kd in the low micromolar(10⁻⁶ M) range or higher, such as more than about any of 1 μM, 2 μM, 3μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM or more. In someembodiments, the sdAb binds its epitope with medium affinity. Amedium-affinity sdAb binds its epitope with a Kd lower than that of alow-affinity sdAb but higher than that of a high-affinity sdAb. In someembodiments, a medium-affinity sdAb binds its epitope with a Kd of anyone of about 1 nM to about 10 nM, about 10 nM to about 100 nM, about 100nM to about 500 nM, about 500 nM to about 1 μM, about 1 nM to about 100nM, about 10 nM to about 500 nM, or about 1 nM to about 1 μM.

In some embodiments, the sdAb has a stronger affinity to its epitopethan the antigen binding portion comprising V_(H) and V_(L). In someembodiments, the sdAb has a weaker affinity to its epitope than theantigen binding portion comprising V_(H) and V_(L). In some embodiments,the difference between the affinity between the sdAb to its epitope andthe antigen binding portion comprising V_(H) and V_(L) and its epitopeis about at least any of 2×, 5×, 10×, 100×, 1000× or more. In someembodiments, the affinity between the sdAb to its epitope is comparableto that between the antigen binding portion comprising V_(H) and V_(L)and its epitope.

In some embodiments, the sdAb specifically binds an immune checkpointmolecule. In some embodiments, the sdAb specifically binds a stimulatoryimmune checkpoint molecule. In some embodiments, the sdAb specificallybinds an inhibitory immune checkpoint molecule. In some embodiments, thesdAb specifically binds an immune checkpoint molecule selected from thegroup consisting of PD-1, PD-L1, PD-L2, CTLA-4, B7-H3, TIM-3, LAG-3,VISTA, ICOS, 4-1BB, OX40, GITR, and CD40. In some embodiments, the sdAbis an agonist for the immune checkpoint molecule. In some embodiments,the sdAb is an antagonist against the immune checkpoint molecule.

Thus, in some embodiments, there is provided a multispecific (such asbispecific) antigen binding protein comprising: (a) a first antigenbinding portion comprising a heavy chain variable domain (V_(H)) and alight chain variable domain (V_(L)), wherein the V_(H) and V_(L)together form an antigen-binding site that specifically binds a firstepitope, and (b) a second antigen binding portion comprising an sdAbthat specifically binds a second epitope of an immune checkpointmolecule, wherein the first antigen binding portion and the secondantigen binding portion are fused to each other. In some embodiments,the sdAb is a camelid, humanized, or human sdAb. In some embodiments,the immune checkpoint molecule is selected from the group consisting ofPD-1, PD-L1, PD-L2, CTLA-4, B7-H3, TIM-3, LAG-3, VISTA, ICOS, 4-1BB,OX40, GITR, and CD40. In some embodiments, the first epitope is from asecond immune checkpoint molecule. In some embodiments, the firstepitope is from a pro-inflammatory molecule, such as a pro-inflammatorycytokine. In some embodiments, the pro-inflammatory molecule is selectedfrom the group consisting of IL-1β, TNF-α, IL-5, IL-6, IL-6R andeotaxin-1. In some embodiments, the first antigen binding portioncomprises a heavy chain comprising the V_(H) and a light chaincomprising the V_(L). In some embodiments, the second antigen bindingportion is fused to the first antigen binding portion at the N-terminusof the heavy chain, the N-terminus of the light chain, the N-terminus ofthe Fc region, the C-terminus of the heavy chain, or the C-terminus ofthe light chain. In some embodiments, the second antigen binding portionis fused to the first antigen binding portion chemically. In someembodiments, the second antigen binding portion is fused to the firstantigen binding portion via a peptide bond or a peptide linker. In someembodiments, the peptide linker is no more than about 30 (such as nomore than about any one of 25, 20, or 15) amino acids long. In someembodiments, the first antigen binding fragment comprises an Fc region,such as an IgG4 Fc.

In some embodiments, the sdAb specifically binds CTLA-4. In someembodiments, the sdAb binds CTLA-4 with high affinity. In someembodiments, the sdAb binds CTLA-4 with medium affinity. In someembodiments, the sdAb binds CTLA-4 with low affinity.

In some embodiments, there is provided a multispecific (such asbispecific) antigen binding protein comprising: (a) a first antigenbinding portion comprising a heavy chain variable domain (V_(H)) and alight chain variable domain (V_(L)), wherein the V_(H) and V_(L)together form an antigen-binding site that specifically binds an epitopeof an immune checkpoint molecule, and (b) a second antigen bindingportion comprising an sdAb (e.g., a V_(H)H) that specifically bindsCTLA-4, wherein the first antigen binding portion and the second antigenbinding portion are fused to each other. In some embodiments, the sdAbis a camelid, humanized, or human sdAb. In some embodiments, the immunecheckpoint molecule is an epitope of CTLA-4 that is different from theepitope specifically recognized by the sdAb. In some embodiments, theimmune checkpoint molecule is selected from the group consisting ofPD-1, PD-L1, PD-L2, B7-H3, TIM-3, LAG-3, VISTA, ICOS, 4-1BB, OX40, GITR,and CD40. In some embodiments, the first antigen binding portioncomprises a full-length anti-PD-1 monoclonal antibody (such aspembrolizumab or nivolumab) or antigen binding fragment thereof. In someembodiments, the first antigen binding portion comprises a full-lengthanti-PD-L1 monoclonal antibody (such as duravalumab or atezolizumab) orantigen binding fragment thereof. In some embodiments, the sdAb bindsCTLA-4 with high affinity. In some embodiments, the sdAb binds CTLA-4with medium affinity. In some embodiments, the sdAb binds CTLA-4 withlow affinity. In some embodiments, the first antigen binding portioncomprises a heavy chain comprising the V_(H) and a light chaincomprising the V_(L). In some embodiments, the second antigen bindingportion is fused to the first antigen binding portion at the N-terminusof the heavy chain, the N-terminus of the light chain, the N-terminus ofthe Fc region, the C-terminus of the heavy chain, or the C-terminus ofthe light chain. In some embodiments, the second antigen binding portionis fused to the first antigen binding portion chemically. In someembodiments, the second antigen binding portion is fused to the firstantigen binding portion via a peptide bond or a peptide linker. In someembodiments, the peptide linker is no more than about 30 (such as nomore than about any one of 25, 20, or 15) amino acids long. In someembodiments, the first antigen binding fragment comprises an Fc region,such as an IgG4 Fc.

In some embodiments, the sdAb specifically binds TIM-3. In someembodiments, the sdAb binds TIM-3 with high affinity. In someembodiments, the sdAb binds TIM-3 with medium affinity. In someembodiments, the sdAb binds TIM-3 with low affinity.

Thus, in some embodiments, there is provided a multispecific (such asbispecific) antigen binding protein comprising: (a) a first antigenbinding portion comprising a heavy chain variable domain (V_(H)) and alight chain variable domain (V_(L)), wherein the V_(H) and V_(L)together form an antigen-binding site that specifically binds an epitopeof an immune checkpoint molecule, and (b) a second antigen bindingportion comprising an sdAb (e.g., a V_(H)H) that specifically bindsTIM-3, wherein the first antigen binding portion and the second antigenbinding portion are fused to each other. In some embodiments, the sdAbis a camelid, humanized, or human sdAb. In some embodiments, the immunecheckpoint molecule is selected from the group consisting of CTLA-4,PD-1, PD-L1, PD-L2, B7-H3, LAG-3, VISTA, ICOS, 4-1BB, OX40, GITR, andCD40. In some embodiments, the first antigen binding portion comprises afull-length anti-PD-1 monoclonal antibody (such as pembrolizumab ornivolumab) or antigen binding fragment thereof. In some embodiments, thefirst antigen binding portion comprises a full-length anti-PD-L1monoclonal antibody (such as duravalumab or atezolizumab) or antigenbinding fragment thereof. In some embodiments, the first antigen bindingportion comprises a heavy chain comprising the V_(H) and a light chaincomprising the V_(L). In some embodiments, the second antigen bindingportion is fused to the first antigen binding portion at the N-terminusof the heavy chain, the N-terminus of the light chain, the N-terminus ofthe Fc region, the C-terminus of the heavy chain, or the C-terminus ofthe light chain. In some embodiments, the second antigen binding portionis fused to the first antigen binding portion chemically. In someembodiments, the second antigen binding portion is fused to the firstantigen binding portion via a peptide bond or a peptide linker. In someembodiments, the peptide linker is no more than about 30 (such as nomore than about any one of 25, 20, or 15) amino acids long. In someembodiments, the first antigen binding fragment comprises an Fc region,such as an IgG4 Fc.

In some embodiments, the sdAb specifically binds LAG-3. In someembodiments, the sdAb binds LAG-3 with high affinity. In someembodiments, the sdAb binds LAG-3 with medium affinity. In someembodiments, the sdAb binds LAG-3 with low affinity.

Thus, in some embodiments, there is provided a multispecific (such asbispecific) antigen binding protein comprising: (a) a first antigenbinding portion comprising a heavy chain variable domain (V_(H)) and alight chain variable domain (V_(L)), wherein the V_(H) and V_(L)together form an antigen-binding site that specifically binds an epitopeof an immune checkpoint molecule, and (b) a second antigen bindingportion comprising an sdAb (e.g., a V_(H)H) that specifically bindsLAG-3, wherein the first antigen binding portion and the second antigenbinding portion are fused to each other. In some embodiments, the sdAbis a camelid, humanized, or human sdAb. In some embodiments, the immunecheckpoint molecule is selected from the group consisting of CTLA-4,PD-1, PD-L1, PD-L2, B7-H3, TIM-3, VISTA, ICOS, 4-1BB, OX40, GITR, andCD40. In some embodiments, the first antigen binding portion comprises afull-length anti-PD-1 monoclonal antibody (such as pembrolizumab ornivolumab) or antigen binding fragment thereof. In some embodiments, thefirst antigen binding portion comprises a full-length anti-PD-L1monoclonal antibody (such as duravalumab or atezolizumab) or antigenbinding fragment thereof. In some embodiments, the first antigen bindingportion comprises a heavy chain comprising the V_(H) and a light chaincomprising the V_(L). In some embodiments, the second antigen bindingportion is fused to the first antigen binding portion at the N-terminusof the heavy chain, the N-terminus of the light chain, the N-terminus ofthe Fc region, the C-terminus of the heavy chain, or the C-terminus ofthe light chain. In some embodiments, the second antigen binding portionis fused to the first antigen binding portion chemically. In someembodiments, the second antigen binding portion is fused to the firstantigen binding portion via a peptide bond or a peptide linker. In someembodiments, the peptide linker is no more than about 30 (such as nomore than about any one of 25, 20, or 15) amino acids long. In someembodiments, the first antigen binding fragment comprises an Fc region,such as an IgG4 Fc.

In some embodiments, the sdAb specifically binds VISTA. In someembodiments, the sdAb binds VISTA with high affinity. In someembodiments, the sdAb binds VISTA with medium affinity. In someembodiments, the sdAb binds VISTA with low affinity.

Thus, in some embodiments, there is provided a multispecific (such asbispecific) antigen binding protein comprising: (a) a first antigenbinding portion comprising a heavy chain variable domain (V_(H)) and alight chain variable domain (V_(L)), wherein the V_(H) and V_(L)together form an antigen-binding site that specifically binds an epitopeof an immune checkpoint molecule, and (b) a second antigen bindingportion comprising an sdAb (e.g., a V_(H)H) that specifically bindsVISTA, wherein the first antigen binding portion and the second antigenbinding portion are fused to each other. In some embodiments, the sdAbis a camelid, humanized, or human sdAb. In some embodiments, the immunecheckpoint molecule is selected from the group consisting of CTLA-4,PD-1, PD-L1, PD-L2, B7-H3, TIM-3, LAG-3, ICOS, 4-1BB, OX40, GITR, andCD40. In some embodiments, the first antigen binding portion comprises afull-length anti-PD-1 monoclonal antibody (such as pembrolizumab ornivolumab) or antigen binding fragment thereof. In some embodiments, thefirst antigen binding portion comprises a full-length anti-PD-L1monoclonal antibody (such as duravalumab or atezolizumab) or antigenbinding fragment thereof. In some embodiments, the first antigen bindingportion comprises a heavy chain comprising the V_(H) and a light chaincomprising the V_(L). In some embodiments, the second antigen bindingportion is fused to the first antigen binding portion at the N-terminusof the heavy chain, the N-terminus of the light chain, the N-terminus ofthe Fc region, the C-terminus of the heavy chain, or the C-terminus ofthe light chain. In some embodiments, the second antigen binding portionis fused to the first antigen binding portion chemically. In someembodiments, the second antigen binding portion is fused to the firstantigen binding portion via a peptide bond or a peptide linker. In someembodiments, the peptide linker is no more than about 30 (such as nomore than about any one of 25, 20, or 15) amino acids long. In someembodiments, the first antigen binding fragment comprises an Fc region,such as an IgG4 Fc.

In some embodiments, the sdAb specifically binds a cell surface antigen.In some embodiments, the cell surface antigen is a tumor antigen. Insome embodiments, the sdAb specifically binds a cell surface antigen onan immune effector cell, such as T cell, or Natural Killer cell.

In some embodiments, there is provided a multispecific (such asbispecific) antigen binding protein comprising: (a) a first antigenbinding portion comprising a heavy chain variable domain (V_(H)) and alight chain variable domain (V_(L)), wherein the V_(H) and V_(L)together form an antigen-binding site that specifically binds a firsttumor antigen, and (b) a second antigen binding portion comprising ansdAb that specifically binds a second tumor antigen, wherein the firstantigen binding portion and the second antigen binding portion are fusedto each other. In some embodiments, the sdAb is a camelid, humanized, orhuman sdAb. In some embodiments, the first tumor antigen and/or thesecond tumor antigen is selected from the group consisting of HER2,BRAF, EGFR, VEGFR2, CD20, RANKL, CD38, and CD52. In some embodiments,the first antigen binding portion comprises a full-length anti-HER-2monoclonal antibody (such as trastuzumab) or antigen binding fragmentthereof. In some embodiments, the first antigen binding portioncomprises a heavy chain comprising the V_(H) and a light chaincomprising the V_(L). In some embodiments, the second antigen bindingportion is fused to the first antigen binding portion at the N-terminusof the heavy chain, the N-terminus of the light chain, the N-terminus ofthe Fc region, the C-terminus of the heavy chain, or the C-terminus ofthe light chain. In some embodiments, the second antigen binding portionis fused to the first antigen binding portion chemically. In someembodiments, the second antigen binding portion is fused to the firstantigen binding portion via a peptide bond or a peptide linker. In someembodiments, the peptide linker is no more than about 30 (such as nomore than about any one of 25, 20, or 15) amino acids long. In someembodiments, the first antigen binding fragment comprises an Fc region,such as an IgG1 Fc.

In some embodiments, the sdAb specifically binds CD3. In someembodiments, the sdAb binds CD3 with high affinity. In some embodiments,the sdAb binds CD3 with medium affinity. In some embodiments, the sdAbbinds CD3 with low affinity.

Thus, in some embodiments, there is provided a multispecific (such asbispecific) antigen binding protein comprising: (a) a first antigenbinding portion comprising a heavy chain variable domain (V_(H)) and alight chain variable domain (V_(L)), wherein the V_(H) and V_(L)together form an antigen-binding site that specifically binds an epitopeof a tumor antigen, and (b) a second antigen binding portion comprisingan sdAb (e.g., a V_(H)H) that specifically binds CD3, wherein the firstantigen binding portion and the second antigen binding portion are fusedto each other. In some embodiments, the sdAb is a camelid, humanized, orhuman sdAb. In some embodiments, the tumor antigen is selected from thegroup consisting of HER2, BRAF, EGFR, VEGFR2, CD20, RANKL, CD38, andCD52. In some embodiments, the first antigen binding portion comprises afull-length anti-HER-2 monoclonal antibody (such as trastuzumab) orantigen binding fragment thereof. In some embodiments, the first antigenbinding portion comprises a heavy chain comprising the V_(H) and a lightchain comprising the V_(L). In some embodiments, the second antigenbinding portion is fused to the first antigen binding portion at theN-terminus of the heavy chain, the N-terminus of the light chain, theN-terminus of the Fc region, the C-terminus of the heavy chain, or theC-terminus of the light chain. In some embodiments, the second antigenbinding portion is fused to the first antigen binding portionchemically. In some embodiments, the second antigen binding portion isfused to the first antigen binding portion via a peptide bond or apeptide linker. In some embodiments, the peptide linker is no more thanabout 30 (such as no more than about any one of 25, 20, or 15) aminoacids long. In some embodiments, the first antigen binding fragmentcomprises an Fc region, such as an IgG4 Fc.

In some embodiments, the sdAb specifically binds an extracellularprotein, such as a secreted protein. In some embodiments, the sdAbspecifically binds a pro-inflammatory molecule. In some embodiments, thesdAb specifically binds an angiogenic factor, such as VEGF.

In some embodiments, the sdAb specifically binds IL-1β. In someembodiments, the sdAb binds IL-1β with high affinity. In someembodiments, the sdAb binds IL-1β with medium affinity. In someembodiments, the sdAb binds IL-1β with low affinity.

Thus, in some embodiments, there is provided a multispecific (such asbispecific) antigen binding protein comprising: (a) a first antigenbinding portion comprising a heavy chain variable domain (V_(H)) and alight chain variable domain (V_(L)), wherein the V_(H) and V_(L)together form an antigen-binding site that specifically binds an epitopeof a pro-inflammatory molecule, and (b) a second antigen binding portioncomprising an sdAb (e.g., a V_(H)H) that specifically binds IL-1β,wherein the first antigen binding portion and the second antigen bindingportion are fused to each other. In some embodiments, the sdAb is acamelid, humanized, or human sdAb. In some embodiments, thepro-inflammatory molecule is selected from the group consisting ofTNF-α, IL-5, IL-6, IL-6R and eotaxin-1. In some embodiments, the firstantigen binding portion comprises a full-length anti-TNF-α monoclonalantibody (such as adalimumab) or antigen binding fragment thereof. Insome embodiments, the first antigen binding portion comprises a heavychain comprising the V_(H) and a light chain comprising the V_(L). Insome embodiments, the second antigen binding portion is fused to thefirst antigen binding portion at the N-terminus of the heavy chain, theN-terminus of the light chain, the N-terminus of the Fc region, theC-terminus of the heavy chain, or the C-terminus of the light chain. Insome embodiments, the second antigen binding portion is fused to thefirst antigen binding portion chemically. In some embodiments, thesecond antigen binding portion is fused to the first antigen bindingportion via a peptide bond or a peptide linker. In some embodiments, thepeptide linker is no more than about 30 (such as no more than about anyone of 25, 20, or 15) amino acids long. In some embodiments, the firstantigen binding fragment comprises an Fc region, such as an IgG1 Fc.

In some embodiments, the sdAb specifically binds eotaxin-1, i.e., CCL11.

Thus, in some embodiments, there is provided a multispecific (such asbispecific) antigen binding protein comprising: (a) a first antigenbinding portion comprising a heavy chain variable domain (V_(H)) and alight chain variable domain (V_(L)), wherein the V_(H) and V_(L)together form an antigen-binding site that specifically binds an epitopeof a pro-inflammatory molecule, and (b) a second antigen binding portioncomprising an sdAb (e.g., a V_(H)H) that specifically binds eotaxin-1,wherein the first antigen binding portion and the second antigen bindingportion are fused to each other. In some embodiments, the sdAb is acamelid, humanized, or human sdAb. In some embodiments, thepro-inflammatory molecule is selected from the group consisting ofIL-1β, TNF-α, IL-5, IL-6 and IL-6R. In some embodiments, the firstantigen binding portion comprises a full-length anti-IL-5 monoclonalantibody (such as mepolizumab) or antigen binding fragment thereof. Insome embodiments, the first antigen binding portion comprises a heavychain comprising the V_(H) and a light chain comprising the V_(L). Insome embodiments, the second antigen binding portion is fused to thefirst antigen binding portion at the N-terminus of the heavy chain, theN-terminus of the light chain, the N-terminus of the Fc region, theC-terminus of the heavy chain, or the C-terminus of the light chain. Insome embodiments, the second antigen binding portion is fused to thefirst antigen binding portion chemically. In some embodiments, thesecond antigen binding portion is fused to the first antigen bindingportion via a peptide bond or a peptide linker. In some embodiments, thepeptide linker is no more than about 30 (such as no more than about anyone of 25, 20, or 15) amino acids long. In some embodiments, the firstantigen binding fragment comprises an Fc region, such as an IgG1 Fc.

Antigen Binding Portion Comprising V_(H) and V_(L)

The MABPs of the present application comprise at least one antigenbinding portion comprising a heavy chain variable domain (V_(H)) and alight chain variable domain (V_(L)). Such antigen binding portion can bea full-length conventional antibody consisting of two heavy chains andtwo light chains, or an antigen binding fragment derived therefrom.

In some embodiments, the first antigen binding portion is an antigenbinding fragment comprising a heavy chain comprising the V_(H) domainand a light chain comprising the V_(L) domain. Exemplary antigen bindingfragments contemplated herein include, but are not limited to, Fab,Fab′, F(ab′)₂, and Fv fragments; diabodies; linear antibodies;single-chain antibody molecules (such as scFv); and multispecificantibodies formed from antibody fragments.

In some embodiments, the first antigen binding portion comprises an Fcregion, such as a human Fc region. In some embodiments, the Fc region isderived from an IgG molecule, such as any one of the IgG1, IgG2, IgG3,or IgG4 subclass. In some embodiments, the Fc region is capable ofmediating an antibody effector function, such as ADCC(antibody-dependent cell-mediated cytotoxicity) and/or CDC(complement-dependent cytotoxicity). For example, antibodies of subclassIgG1, IgG2, and IgG3 with wildtype Fc sequences usually show complementactivation including CIq and C3 binding, whereas IgG4 does not activatethe complement system and does not bind CIq and/or C3. In someembodiments, the Fc region comprises a modification that reduces bindingaffinity of the Fc region to an Fc receptor. In some embodiments, the Fcregion is an IgG1 Fc. In some embodiments, the IgG1 Fc comprises one ormutations in positions 233-236, such as L234A and/or L235A. In someembodiments, the Fc region is an IgG4 Fc. In some embodiments, the IgG4Fc comprises a mutation in positions 327, 330 and/or 331. See, forexample, Armour K L et al., Eru. J. Immunol. 1999; 29: 2613; and ShieldsR L et al., J. Biol. Chem. 2001; 276: 6591. In some embodiments, the Fcregion comprises a P329G mutation.

In some embodiments, the Fc region comprises a modification thatpromotes heterodimerization of two non-identical heavy chains. Suchmodified Fc regions may be of particular interest for MABPs describedherein having an asymmetric design. In some embodiments, saidmodification is a knob-into-hole modification, comprising a knobmodification in one of the heavy chains or heavy chain fusionpolypeptides and a hole modification in the other one of the two heavychains or heavy chain fusion polypeptides. In one embodiment, the Fcregion comprises a modification within the interface between the twoheavy chains in the CH3 domain, wherein i) in the CH3 domain of oneheavy chain, an amino acid residue is replaced with an amino acidresidue having a larger side chain volume, thereby generating aprotuberance (“knob”) within the interface in the CH3 domain of oneheavy chain which is positionable in a cavity (“hole”) within theinterface in the CH3 domain of the other heavy chain, and ii) in the CH3domain of the other heavy chain, an amino acid residue is replaced withan amino acid residue having a smaller side chain volume, therebygenerating a cavity (“hole”) within the interface in the second CH3domain within which a protuberance (“knob”) within the interface in thefirst CH3 domain is positionable. Examples of knob-into-holemodifications have been described, for example, in US 2011/0287009,US2007/0178552, WO 96/027011, WO 98/050431, and Zhu et al., 1997,Protein Science 6:781-788. Other modifications to the Fc region thatpromote heterodimerization are also contemplated herein. For example,electrostatic steering effects can be engineered into the Fc region toprovide Fc-heterodimeric molecules (see, e.g., U.S. Pat. No. 4,676,980,and Brennan et al., Science, 229: 81 (1985)).

In some embodiments, the Fc region comprises a modification thatinhibits Fab arm exchange. For example, the S228P mutation in IgG4 Fcprevents Fab arm exchange.

In some embodiments, the first antigen binding portion comprises a kappalight chain constant region. In some embodiments, the first antigenbinding portion comprises a lambda light chain constant region. In someembodiments, the first antigen binding portion comprises a light chainconstant region comprising the amino acid sequence of SEQ ID NO: 6.

In some embodiments, the first antigen binding portion comprises a heavychain constant region comprising the amino acid sequence of SEQ ID NO:7.

In some embodiments, the first antigen binding portion is a full-lengthantibody consisting of two heavy chains and two light chains. In someembodiments, the first antigen binding portion comprises a monoclonalantibody consisting of two heavy chains and two light chains (alsoreferred herein as “4-chain antibody”). In some embodiments, the firstantigen binding portion comprises a multispecific (such as bispecific)full-length antibody consisting of two heavy chains and two lightchains. In some embodiments, the first antigen binding portion comprisesa full-length antibody of human IgG1 subclass, or of human IgG1 subclasswith the mutations L234A and L235A. In some embodiments, the firstantigen binding portion comprises a full-length antibody of human IgG2subclass. In some embodiments, the first antigen binding portioncomprises a full-length antibody of human IgG3 subclass. In someembodiments, the first antigen binding portion comprises a full-lengthantibody of human IgG4 subclass or, of human IgG4 subclass with theadditional mutation S228P.

Any full-length 4-chain antibody known in the art or antigen bindingfragments derived therefrom can be used as the first antigen bindingportion in the MABP of the present application. Antibodies or antibodyfragments with proven clinical efficacy, safety, and pharmacokineticsprofile are of particular interest. In some embodiments, the antibody orantibody fragment known in the art is further engineered, such ashumanized or mutagenized to select for a variant with a suitableaffinity, prior to fusion with the second antigen binding portion toprovide the MABP. In some embodiments, the first antigen binding portioncomprises the V_(H) and V_(L) domains of a monoclonal antibody orantibody fragment known in the art, and modified heavy chain constantregion and/or light chain constant region. In some embodiments, thefirst antigen binding portion comprises the monoclonal antibody known inthe art and a modified Fc region, such as an IgG4 Fc with an S228Pmutation. In some embodiments, the first antigen binding portioncomprises a human, humanized, or chimeric full-length antibody orantibody fragments.

In some embodiments, the first antigen binding portion is derived froman approved (such as by FDA and/or EMA) or investigational monoclonalantibody or antibody fragment (such as Fab). In some embodiments, thefirst antigen binding portion is an approved (such as by FDA and/or EMA)or investigational monoclonal antibody or antibody fragment (such asFab).

In some embodiments, the first antigen binding portion specificallybinds an immune checkpoint molecule. In some embodiments, the firstantigen binding portion comprises a full-length antibody (such asantagonist antibody) or antigen binding fragment derived therefrom thatspecifically binds an inhibitory immune checkpoint protein. In someembodiments, the first antigen binding portion comprises a full-lengthantibody (such as agonist antibody) or antigen binding fragment derivedtherefrom that specifically binds a stimulatory checkpoint molecule. Insome embodiments, the immune checkpoint molecule is selected from thegroup consisting of PD-1, PD-L1, PD-L2, CTLA-4, B7-H3, TIM-3, LAG-3,VISTA, ICOS, 4-1BB, OX40, GITR, and CD40. In some embodiments, the firstantigen binding portion is an anti-PD-1 antibody or antigen bindingfragment thereof. In some embodiments, the anti-PD-1 antibody isselected from the group consisting of pembrolizumab and nivolumab. Insome embodiments, the first antigen binding portion is an anti-PD-L1antibody or antigen binding fragment thereof. In some embodiments, theanti-PD-L1 antibody is duravalumab or atezolizumab. In some embodiments,the first antigen binding portion is an anti-CTLA-4 antibody or antigenbinding fragment thereof. In some embodiments, the anti-CTLA-4 antibodyis ipilimumab.

In some embodiments, the first antigen binding portion comprisespembrolizumab or antigen binding fragment thereof. In some embodiments,the first antigen binding portion comprises a V_(H) domain comprisingthe amino acid sequence of SEQ ID NO: 2 and a V_(L) domain comprisingthe amino acid sequence of SEQ ID NO: 3. In some embodiments, the firstantigen binding portion comprises an IgG4 Fc. In some embodiments, thefirst antigen binding portion comprises a heavy chain comprising theamino acid sequence of SEQ ID NO: 4. In some embodiments, the firstantigen binding portion comprises a light chain comprising the aminoacid sequence of SEQ ID NO: 5. In some embodiments, the first antigenbinding portion comprises an IgG4 Fc.

Pembrolizumab (e.g., KEYTRUDA®) is a humanized antibody used in cancerimmunotherapy. It targets the programmed cell death 1 (PD-1) receptor.The drug was initially used in treating metastatic melanoma. On Sep. 4,2014 the US Food and Drug Administration (FDA) approved KEYTRUDA® underthe FDA Fast Track Development Program. It is approved for use inadvanced melanoma. On Oct. 2, 2015, the US FDA approved KEYTRUDA® forthe treatment of metastatic non-small cell lung cancer in patients whosetumors express PD-L1 and who have failed treatments with otherchemotherapeutic agents.

Ipilimumab (e.g., YERVOY) is a fully human anti-CTLA-4 immunoglobulin G1(IgG1) monoclonal antibody (mAb) that blocks the down-regulation ofT-cell activation. Ipilimumab is a CTLA-4 immune checkpoint inhibitorthat blocks T-cell inhibitory signals induced by the CTLA-4 pathway, andincreases the number of tumor reactive T effector cells. Ipilimumab wasused in combination with nivolumab (e.g., OPDIVO®) to investigate theeffects of concurrent inhibition of the PD-1 and CTLA-4 receptors innonhuman primates. OPDIVO® has demonstrated clinical efficacy either asmonotherapy or in combination with ipilimumab in treating several tumortypes, including renal cell carcinoma, melanoma, NSCLC, and somelymphomas. BMS recently announced the treatment results of immunecombination therapy OPDIVO® and ipilimumab for treating melanoma.Compared with ipilimumab monotherapy, the combined therapy achieved avery high objective response rate (61% vs 11%) and complete remissionrate of 22%, while disease progression or death risk decreased by 60%.This kind of therapy demonstrated the great potential of differentcombinations of immune therapeutic agents in clinical treatment ofcancer.

In some embodiments, the first antigen binding portion specificallybinds a tumor antigen. In some embodiments, the tumor antigen isselected from the group consisting of HER2, BRAF, EGFR, VEGFR2, CD20,RANKL, CD38, and CD52. In some embodiments, the first antigen bindingportion is an anti-HER2 antibody or antigen binding fragment thereof. Insome embodiments, the anti-HER2 antibody is trastuzumab.

Trastuzumab (HERCEPTIN®), one of the five top selling therapeuticantibodies, is a humanized anti-HER2 receptor monoclonal antibody thathas significantly increased the survival rate in patients withHER2-positive breast cancer. The HER receptors are proteins that areembedded in the cell membrane and communicate molecular signals fromoutside the cell (molecules called EGFs) to inside the cell, and turngenes on and off. The HER protein, Human Epidermal Growth FactorReceptor, binds Human Epidermal Growth Factor, and stimulates cellproliferation. In some cancers, notably certain types of breast cancer,HER2 is over-expressed, and causes cancer cells to reproduceuncontrollably. However, among breast cancer patients, only 15-20% ofthem exhibit amplification and overexpression of the human epidermalgrowth factor receptor 2 (HER2), most HER2-patients do not respond totrastuzumab. In addition, some of the HER2+ patients have developedresistance to trastuzumab after initial treatment. As the epidermalgrowth factor RTK family consists of four members: EGFR, HER2, HER3 andHER4, some bispecific antibodies have been developed to target two ofthese antigens, which have shown advantages over conventionalmonospecific antibodies.

In some embodiments, the first antigen binding portion specificallybinds an angiogenic factor. In some embodiments, the first antigenbinding portion is an anti-Ang2 antibody or antigen binding fragmentthereof, such as LC10.

In some embodiments, the first antigen binding portion specificallybinds a pro-inflammatory molecule. In some embodiments, thepro-inflammatory molecule is selected from the group consisting ofIL-1β, TNF-α, IL-5, IL-6, IL-6R and eotaxin-1. In some embodiments, thefirst antigen binding portion is an anti-TNF-α antibody or antigenbinding fragment thereof. In some embodiments, the anti-TNF-α antibodyis adalimumab.

Exemplary Multispecific Antigen Binding Proteins

In some embodiments, there is provided a multispecific (such asbispecific) antigen binding protein comprising: (a) a first antigenbinding portion comprising a full-length antibody (such as pembrolizumabor nivolumab) consisting of two heavy chains and two light chains,wherein the full-length antibody specifically binds PD-1; and (b) asecond antigen binding portion comprising an sdAb that specificallybinds CTLA-4, wherein the first antigen binding portion and the secondantigen binding portion are fused to each other. In some embodiments,the sdAb is a camelid, humanized, or human sdAb. In some embodiments,the sdAb binds CTLA-4 with a high affinity. In some embodiments, thesdAb binds CTLA-4 with a medium affinity. In some embodiments, the sdAbbinds CTLA-4 with a low affinity. In some embodiments, the secondantigen binding portion is fused to the first antigen binding portion atthe N-terminus of one or each of the two heavy chains, the N-terminus ofone or each of the two light chains, the N-terminus of the Fc region,the C-terminus of one or each of the two heavy chains, or the C-terminusof one or each of the two light chains. In some embodiments, the secondantigen binding portion is fused to the first antigen binding portionchemically. In some embodiments, the second antigen binding portion isfused to the first antigen binding portion via a peptide bond or apeptide linker. In some embodiments, the peptide linker is no more thanabout 30 (such as no more than about any one of 25, 20, or 15) aminoacids long. In some embodiments, the peptide linker comprises the aminoacid sequence of SEQ ID NO: 1, 8 or 13. In some embodiments, the firstantigen binding fragment comprises an Fc region, such as an IgG4 Fc.

In some embodiments, there is provided a bispecific antigen bindingprotein comprising: (a) a first polypeptide comprising from N-terminusto C-terminus: V_(H)-C_(H)1-C_(H)2-C_(H)3-V_(H)H; and (b) a secondpolypeptide comprising from N-terminus to C-terminus: VL-CL, whereinV_(H) and V_(L) forms an antigen binding site that specifically bindsPD-1, and wherein V_(H)H specifically binds CTLA-4. In some embodiments,the V_(H) and V_(L) domains are derived from pembrolizumab or nivolumab.In some embodiments, the C_(H)3 and V_(H)H domains are fused to eachother via a peptide linker, such as a peptide linker comprising theamino acid sequence of SEQ ID NO: 1, 8 or 13. In some embodiments, theC_(H)2 and C_(H)3 domains are derived from an IgG4 Fc. In someembodiments, the BABP has the structure as shown in FIG. 4 .

In some embodiments, there is provided a bispecific antigen bindingprotein comprising: (a) a first polypeptide comprising from N-terminusto C-terminus: V_(H)H-V_(H)-C_(H)1-C_(H)2-C_(H)3; and (b) a secondpolypeptide comprising from N-terminus to C-terminus: VL-CL, whereinV_(H) and V_(L) forms an antigen binding site that specifically bindsPD-1, and wherein V_(H)H specifically binds CTLA-4. In some embodiments,the V_(H) and V_(L) domains are derived from pembrolizumab or nivolumab.In some embodiments, the V_(H) and V_(H)H domains are fused to eachother via a peptide linker, such as a peptide linker comprising theamino acid sequence of SEQ ID NO: 1, 8 or 13. In some embodiments, theC_(H)2 and C_(H)3 domains are derived from an IgG4 Fc. In someembodiments, the BABP has the structure as shown in FIG. 9 .

In some embodiments, there is provided a bispecific antigen bindingprotein comprising: (a) a first polypeptide comprising from N-terminusto C-terminus: V_(H)-C_(H)1-C_(H)2-C_(H)3; and (b) a second polypeptidecomprising from N-terminus to C-terminus: VL-CL-V_(H)H, wherein V_(H)and V_(L) forms an antigen binding site that specifically binds PD-1,and wherein V_(H)H specifically binds CTLA-4. In some embodiments, theV_(H) and V_(L) domains are derived from pembrolizumab or nivolumab. Insome embodiments, the C_(L) and V_(H)H domains are fused to each othervia a peptide linker, such as a peptide linker comprising the amino acidsequence of SEQ ID NO: 1, 8 or 13. In some embodiments, the C_(H)2 andC_(H)3 domains are derived from an IgG4 Fc. In some embodiments, theBABP has the structure as shown in FIG. 11 .

In some embodiments, there is provided a bispecific antigen bindingprotein comprising: (a) a first polypeptide comprising from N-terminusto C-terminus: V_(H)—C_(H)1-C_(H)2-C_(H)3; and (b) a second polypeptidecomprising from N-terminus to C-terminus: V_(H)H-VL-CL, wherein V_(H)and V_(L) forms an antigen binding site that specifically binds PD-1,and wherein V_(H)H specifically binds CTLA-4. In some embodiments, theV_(H) and V_(L) domains are derived from pembrolizumab or nivolumab. Insome embodiments, the V_(L) and V_(H)H domains are fused to each othervia a peptide linker, such as a peptide linker comprising the amino acidsequence of SEQ ID NO: 1, 8 or 13. In some embodiments, the C_(H)2 andC_(H)3 domains are derived from an IgG4 Fc. In some embodiments, theBABP has the structure as shown in FIG. 13 .

In some embodiments, there is provided a bispecific antigen bindingprotein comprising: (a) a first polypeptide comprising from N-terminusto C-terminus: V_(H)H-V_(H)-C_(H)1-C_(H)2-C_(H)3; and (b) a secondpolypeptide comprising from N-terminus to C-terminus: V_(H)H-VL-CL,wherein V_(H) and V_(L) forms an antigen binding site that specificallybinds PD-1, and wherein V_(H)H specifically binds CTLA-4. In someembodiments, the V_(H) and V_(L) domains are derived from pembrolizumabor nivolumab. In some embodiments, the V_(L) and V_(H)H domains, and/orthe V_(L) and V_(H)H domains are fused to each other via a peptidelinker, such as a peptide linker comprising the amino acid sequence ofSEQ ID NO: 1, 8 or 13. In some embodiments, the C_(H)2 and C_(H)3domains are derived from an IgG4 Fc. In some embodiments, the BABP hasthe structure as shown in FIG. 17 .

In some embodiments, there is provided a bispecific antigen bindingprotein comprising: (a) a first polypeptide comprising from N-terminusto C-terminus: V_(H)H1-V_(H)H2-V_(H)-C_(H)1-C_(H)2-C_(H)3; and (b) asecond polypeptide comprising from N-terminus to C-terminus: VL-CL,wherein V_(H) and V_(L) forms an antigen binding site that specificallybinds PD-1, and wherein V_(H)H specifically binds CTLA-4. In someembodiments, the V_(H) and V_(L) domains are derived from pembrolizumabor nivolumab. In some embodiments, the V_(H)H1 and V_(H)H2 domains,and/or the V_(H) and V_(H)H2 domains are fused to each other via apeptide linker, such as a peptide linker comprising the amino acidsequence of SEQ ID NO: 1, 8 or 13. In some embodiments, the C_(H)2 andC_(H)3 domains are derived from an IgG4 Fc. In some embodiments, theBABP has the structure as shown in FIG. 18 .

In some embodiments, there is provided a bispecific antigen bindingprotein comprising: (a) a first polypeptide comprising from N-terminusto C-terminus: V_(H)—C_(H)1-V_(H)H-C_(H)2-C_(H)3; and (b) a secondpolypeptide comprising from N-terminus to C-terminus: VL-CL, whereinV_(H) and V_(L) forms an antigen binding site that specifically bindsPD-1, and wherein V_(H)H specifically binds CTLA-4. In some embodiments,the V_(H) and V_(L) domains are derived from pembrolizumab or nivolumab.In some embodiments, the C_(H)1 and V_(H)H domains are fused to eachother via a peptide linker, such as a peptide linker comprising theamino acid sequence of SEQ ID NO: 1, 8 or 13. In some embodiments, theC_(H)2 and C_(H)3 domains are derived from an IgG4 Fc. In someembodiments, the BABP has the structure as shown in FIG. 19 .

In some embodiments, there is provided a bispecific antigen bindingprotein comprising a polypeptide comprising from N-terminus toC-terminus: scFv-V_(H)H-C_(H)2-C_(H)3, wherein the scFv thatspecifically binds PD-1, and wherein V_(H)H specifically binds CTLA-4.In some embodiments, the scFv derived from pembrolizumab or nivolumab.In some embodiments, the scFv and V_(H)H domains are fused to each othervia a peptide linker, such as a peptide linker comprising the amino acidsequence of SEQ ID NO: 1, 8 or 13. In some embodiments, the C_(H)2 andC_(H)3 domains are derived from an IgG4 Fc. In some embodiments, theBABP has the structure as shown in FIG. 20 .

In some embodiments, there is provided a bispecific antigen bindingprotein comprising: (a) a first polypeptide comprising from N-terminusto C-terminus: V_(H)—C_(H)1-V_(H)H-C_(H)1-C_(H)2-C_(H)3; and (b) asecond polypeptide comprising from N-terminus to C-terminus:V_(L)—C_(L)-V_(H)H-C_(L), wherein V_(H) and V_(L) forms an antigenbinding site that specifically binds PD-1, and wherein V_(H)Hspecifically binds CTLA-4. In some embodiments, the V_(H) and V_(L)domains are derived from pembrolizumab or nivolumab. In someembodiments, the C_(H)1 and V_(H)H domains, and/or C_(L) and V_(H)Hdomains are fused to each other via a peptide linker, such as a peptidelinker comprising the amino acid sequence of SEQ ID NO: 1, 8 or 13. Insome embodiments, the C_(H)2 and C_(H)3 domains are derived from an IgG4Fc. In some embodiments, the BABP has the structure as shown in FIG. 21.

In some embodiments, there is provided a bispecific antigen bindingprotein comprising: (a) a first polypeptide comprising from N-terminusto C-terminus: scFv-V_(H)H-C_(H)2-C_(H)3; and (b) a second polypeptidecomprising from N-terminus to C-terminus: V_(H)H-C_(L), wherein the scFvspecifically binds PD-1, and wherein V_(H)H specifically binds CTLA-4.In some embodiments, the scFv is derived from pembrolizumab ornivolumab. In some embodiments, the scFv and V_(H)H domains are fused toeach other via a peptide linker, such as a peptide linker comprising theamino acid sequence of SEQ ID NO: 1, 8 or 13. In some embodiments, theC_(H)2 and C_(H)3 domains are derived from an IgG4 Fc. In someembodiments, the BABP has the structure as shown in FIG. 22 .

In some embodiments, there is provided a multispecific (such asbispecific) antigen binding protein comprising: (a) a first antigenbinding portion comprising a full-length antibody (such as pembrolizumabor nivolumab) consisting of two heavy chains and two light chains,wherein the full-length antibody specifically binds PD-1; and (b) asecond antigen binding portion comprising an sdAb that specificallybinds TIM-3, wherein the first antigen binding portion and the secondantigen binding portion are fused to each other. In some embodiments,the sdAb is a camelid, humanized, or human sdAb. In some embodiments,the second antigen binding portion is fused to the first antigen bindingportion at the N-terminus of one or each of the two heavy chains, theN-terminus of one or each of the two light chains, the N-terminus of theFc region, the C-terminus of one or each of the two heavy chains, or theC-terminus of one or each of the two light chains. In some embodiments,the second antigen binding portion is fused to the first antigen bindingportion chemically. In some embodiments, the second antigen bindingportion is fused to the first antigen binding portion via a peptide bondor a peptide linker. In some embodiments, the peptide linker is no morethan about 30 (such as no more than about any one of 25, 20, or 15)amino acids long. In some embodiments, the peptide linker comprises theamino acid sequence of SEQ ID NO: 1, 8 or 13. In some embodiments, thefirst antigen binding fragment comprises an Fc region, such as an IgG4Fc.

In some embodiments, there is provided a multispecific (such asbispecific) antigen binding protein comprising: (a) a first antigenbinding portion comprising a full-length antibody (such as pembrolizumabor nivolumab) consisting of two heavy chains and two light chains,wherein the full-length antibody specifically binds PD-1; and (b) asecond antigen binding portion comprising an sdAb that specificallybinds LAG-3, wherein the first antigen binding portion and the secondantigen binding portion are fused to each other. In some embodiments,the sdAb is a camelid, humanized, or human sdAb. In some embodiments,the second antigen binding portion is fused to the first antigen bindingportion at the N-terminus of one or each of the two heavy chains, theN-terminus of one or each of the two light chains, the N-terminus of theFc region, the C-terminus of one or each of the two heavy chains, or theC-terminus of one or each of the two light chains. In some embodiments,the second antigen binding portion is fused to the first antigen bindingportion chemically. In some embodiments, the second antigen bindingportion is fused to the first antigen binding portion via a peptide bondor a peptide linker. In some embodiments, the peptide linker is no morethan about 30 (such as no more than about any one of 25, 20, or 15)amino acids long. In some embodiments, the peptide linker comprises theamino acid sequence of SEQ ID NO: 1, 8 or 13. In some embodiments, thefirst antigen binding fragment comprises an Fc region, such as an IgG4Fc.

In some embodiments, there is provided a multispecific (such asbispecific) antigen binding protein comprising: (a) a first antigenbinding portion comprising a full-length antibody (such as pembrolizumabor nivolumab) consisting of two heavy chains and two light chains,wherein the full-length antibody specifically binds PD-1; and (b) asecond antigen binding portion comprising an sdAb that specificallybinds VISTA, wherein the first antigen binding portion and the secondantigen binding portion are fused to each other. In some embodiments,the sdAb is a camelid, humanized, or human sdAb. In some embodiments,the second antigen binding portion is fused to the first antigen bindingportion at the N-terminus of one or each of the two heavy chains, theN-terminus of one or each of the two light chains, the N-terminus of theFc region, the C-terminus of one or each of the two heavy chains, or theC-terminus of one or each of the two light chains. In some embodiments,the second antigen binding portion is fused to the first antigen bindingportion chemically. In some embodiments, the second antigen bindingportion is fused to the first antigen binding portion via a peptide bondor a peptide linker. In some embodiments, the peptide linker is no morethan about 30 (such as no more than about any one of 25, 20, or 15)amino acids long. In some embodiments, the peptide linker comprises theamino acid sequence of SEQ ID NO: 1, 8 or 13. In some embodiments, thefirst antigen binding fragment comprises an Fc region, such as an IgG4Fc.

In some embodiments, there is provided a multispecific (such asbispecific) antigen binding protein comprising: (a) a first antigenbinding portion comprising a heavy chain comprising a V_(H) domaincomprising the amino acid sequence of SEQ ID NO: 2 and a light chaincomprising a V_(L) domain comprising the amino acid sequence of SEQ IDNO: 3; and (b) a second antigen binding portion comprising ananti-CTLA-4 sdAb, and wherein the first antigen binding portion and thesecond antigen binding portion are fused to each other. In someembodiments, the sdAb is a camelid, humanized, or human sdAb. In someembodiments, the first antigen binding portion is full-lengthpembrolizumab. In some embodiments, the N-terminus of the second antigenbinding portion is fused to the C-terminus of the heavy chain of thefirst antigen binding portion via an optional peptide linker. In someembodiments, the C-terminus of the second antigen binding portion isfused to the N-terminus of the heavy chain of the first antigen bindingportion via an optional peptide linker. In some embodiments, the peptidelinker is no more than about 30 (such as no more than about any one of25, 20, or 15) amino acids long. In some embodiments, the peptide linkercomprises the amino acid sequence of SEQ ID NO: 1, 8 or 13. In someembodiments, the first antigen binding fragment comprises an Fc region,such as an IgG4 Fc.

In some embodiments, there is provided a multispecific (such asbispecific) antigen binding protein comprising: (a) a first antigenbinding portion comprising a full-length antibody (such as duravalumabor atezolizumab) consisting of two heavy chains and two light chains,wherein the full-length antibody specifically binds PD-L1; and (b) asecond antigen binding portion comprising an sdAb that specificallybinds CTLA-4, wherein the first antigen binding portion and the secondantigen binding portion are fused to each other. In some embodiments,the sdAb is a camelid, humanized, or human sdAb. In some embodiments,the sdAb binds CTLA-4 with a high affinity. In some embodiments, thesdAb binds CTLA-4 with a medium affinity. In some embodiments, the sdAbbinds CTLA-4 with a low affinity. In some embodiments, the secondantigen binding portion is fused to the first antigen binding portion atthe N-terminus of one or each of the two heavy chains, the N-terminus ofone or each of the two light chains, the N-terminus of the Fc region,the C-terminus of one or each of the two heavy chains, or the C-terminusof one or each of the two light chains. In some embodiments, the secondantigen binding portion is fused to the first antigen binding portionchemically. In some embodiments, the second antigen binding portion isfused to the first antigen binding portion via a peptide bond or apeptide linker. In some embodiments, the peptide linker is no more thanabout 30 (such as no more than about any one of 25, 20, or 15) aminoacids long. In some embodiments, the peptide linker comprises the aminoacid sequence of SEQ ID NO: 1, 8 or 13. In some embodiments, the firstantigen binding fragment comprises an Fc region, such as an IgG4 Fc.

In some embodiments, there is provided a bispecific antigen bindingprotein comprising: (a) a first polypeptide comprising from N-terminusto C-terminus: V_(H)H-V_(H)-C_(H)1-C_(H)2-C_(H)3; and (b) a secondpolypeptide comprising from N-terminus to C-terminus: VL-CL, whereinV_(H) and V_(L) forms an antigen binding site that specifically bindsPD-L1, and wherein V_(H)H specifically binds CTLA-4. In someembodiments, the V_(H) and V_(L) domains are derived from atezolizumab.In some embodiments, the V_(H) and V_(H)H domains are fused to eachother via a peptide linker, such as a peptide linker comprising theamino acid sequence of SEQ ID NO: 1, 8 or 13. In some embodiments, theC_(H)2 and C_(H)3 domains are derived from an IgG4 Fc. In someembodiments, the BABP has the structure as shown in FIG. 9 .

In some embodiments, there is provided a multispecific (such asbispecific) antigen binding protein comprising: (a) a first antigenbinding portion comprising a full-length antibody (such as duravalumabor atezolizumab) consisting of two heavy chains and two light chains,wherein the full-length antibody specifically binds PD-L1; and (b) asecond antigen binding portion comprising an sdAb that specificallybinds TIM-3, wherein the first antigen binding portion and the secondantigen binding portion are fused to each other. In some embodiments,the sdAb is a camelid, humanized, or human sdAb. In some embodiments,the second antigen binding portion is fused to the first antigen bindingportion at the N-terminus of one or each of the two heavy chains, theN-terminus of one or each of the two light chains, the N-terminus of theFc region, the C-terminus of one or each of the two heavy chains, or theC-terminus of one or each of the two light chains. In some embodiments,the second antigen binding portion is fused to the first antigen bindingportion chemically. In some embodiments, the second antigen bindingportion is fused to the first antigen binding portion via a peptide bondor a peptide linker. In some embodiments, the peptide linker is no morethan about 30 (such as no more than about any one of 25, 20, or 15)amino acids long. In some embodiments, the peptide linker comprises theamino acid sequence of SEQ ID NO: 1, 8 or 13. In some embodiments, thefirst antigen binding fragment comprises an Fc region, such as an IgG4Fc.

In some embodiments, there is provided a multispecific (such asbispecific) antigen binding protein comprising: (a) a first antigenbinding portion comprising a full-length antibody (such as duravalumabor atezolizumab) consisting of two heavy chains and two light chains,wherein the full-length antibody specifically binds PD-L1; and (b) asecond antigen binding portion comprising an sdAb that specificallybinds LAG-3, wherein the first antigen binding portion and the secondantigen binding portion are fused to each other. In some embodiments,the sdAb is a camelid, humanized, or human sdAb. In some embodiments,the second antigen binding portion is fused to the first antigen bindingportion at the N-terminus of one or each of the two heavy chains, theN-terminus of one or each of the two light chains, the N-terminus of theFc region, the C-terminus of one or each of the two heavy chains, or theC-terminus of one or each of the two light chains. In some embodiments,the second antigen binding portion is fused to the first antigen bindingportion chemically. In some embodiments, the second antigen bindingportion is fused to the first antigen binding portion via a peptide bondor a peptide linker. In some embodiments, the peptide linker is no morethan about 30 (such as no more than about any one of 25, 20, or 15)amino acids long. In some embodiments, the peptide linker comprises theamino acid sequence of SEQ ID NO: 1, 8 or 13. In some embodiments, thefirst antigen binding fragment comprises an Fc region, such as an IgG4Fc.

In some embodiments, there is provided a multispecific (such asbispecific) antigen binding protein comprising: (a) a first antigenbinding portion comprising a full-length antibody (such as duravalumabor atezolizumab) consisting of two heavy chains and two light chains,wherein the full-length antibody specifically binds PD-L1; and (b) asecond antigen binding portion comprising an sdAb that specificallybinds VISTA, wherein the first antigen binding portion and the secondantigen binding portion are fused to each other. In some embodiments,the sdAb is a camelid, humanized, or human sdAb. In some embodiments,the second antigen binding portion is fused to the first antigen bindingportion at the N-terminus of one or each of the two heavy chains, theN-terminus of one or each of the two light chains, the N-terminus of theFc region, the C-terminus of one or each of the two heavy chains, or theC-terminus of one or each of the two light chains. In some embodiments,the second antigen binding portion is fused to the first antigen bindingportion chemically. In some embodiments, the second antigen bindingportion is fused to the first antigen binding portion via a peptide bondor a peptide linker. In some embodiments, the peptide linker is no morethan about 30 (such as no more than about any one of 25, 20, or 15)amino acids long. In some embodiments, the peptide linker comprises theamino acid sequence of SEQ ID NO: 1, 8 or 13. In some embodiments, thefirst antigen binding fragment comprises an Fc region, such as an IgG4Fc.

In some embodiments, there is provided a multispecific (such asbispecific) antigen binding protein comprising: (a) a first antigenbinding portion comprising a full-length antibody (such as trastuzumab)consisting of two heavy chains and two light chains, wherein thefull-length antibody specifically binds HER2 receptor; and (b) a secondantigen binding portion comprising an sdAb that specifically binds CD3,wherein the first antigen binding portion and the second antigen bindingportion are fused to each other. In some embodiments, the sdAb is acamelid, humanized, or human sdAb. In some embodiments, the secondantigen binding portion is fused to the first antigen binding portion atthe N-terminus of one or each of the two heavy chains, the N-terminus ofone or each of the two light chains, the N-terminus of the Fc region,the C-terminus of one or each of the two heavy chains, or the C-terminusof one or each of the two light chains. In some embodiments, the secondantigen binding portion is fused to the first antigen binding portionchemically. In some embodiments, the second antigen binding portion isfused to the first antigen binding portion via a peptide bond or apeptide linker. In some embodiments, the peptide linker is no more thanabout 30 (such as no more than about any one of 25, 20, or 15) aminoacids long. In some embodiments, the peptide linker comprises the aminoacid sequence of SEQ ID NO: 1, 8 or 13. In some embodiments, the firstantigen binding fragment comprises an Fc region, such as IgG4 Fc.

In some embodiments, there is provided a multispecific (such asbispecific) antigen binding protein comprising: (a) a first antigenbinding portion comprising a full-length antibody (such as LC10)consisting of two heavy chains and two light chains, wherein thefull-length antibody specifically binds Ang2; and (b) a second antigenbinding portion comprising an sdAb that specifically binds VEGF, whereinthe first antigen binding portion and the second antigen binding portionare fused to each other. In some embodiments, the sdAb is a camelid,humanized, or human sdAb. In some embodiments, the second antigenbinding portion is fused to the first antigen binding portion at theN-terminus of one or each of the two heavy chains, the N-terminus of oneor each of the two light chains, the N-terminus of the Fc region, theC-terminus of one or each of the two heavy chains, or the C-terminus ofone or each of the two light chains. In some embodiments, the secondantigen binding portion is fused to the first antigen binding portionchemically. In some embodiments, the second antigen binding portion isfused to the first antigen binding portion via a peptide bond or apeptide linker. In some embodiments, the peptide linker is no more thanabout 30 (such as no more than about any one of 25, 20, or 15) aminoacids long. In some embodiments, the peptide linker comprises the aminoacid sequence of SEQ ID NO: 1, 8 or 13. In some embodiments, the firstantigen binding fragment comprises an Fc region, such as IgG1 Fc.

In some embodiments, there is provided a bispecific antigen bindingprotein comprising: (a) a first polypeptide comprising from N-terminusto C-terminus: V_(H)-C_(H)1-C_(H)2-C_(H)3-V_(H)H; and (b) a secondpolypeptide comprising from N-terminus to C-terminus: VL-CL, whereinV_(H) and V_(L) forms an antigen binding site that specifically bindsAng2, and wherein V_(H)H specifically binds VEGF. In some embodiments,the V_(H) and V_(L) domains are derived from LC10. In some embodiments,the C_(H)3 and V_(H)H domains are fused to each other via a peptidelinker, such as a peptide linker comprising the amino acid sequence ofSEQ ID NO: 1, 8 or 13. In some embodiments, the C_(H)2 and C_(H)3domains are derived from an IgG1 Fc. In some embodiments, the BABP hasthe structure as shown in FIG. 4 .

In some embodiments, there is provided a bispecific antigen bindingprotein comprising: (a) a first polypeptide comprising from N-terminusto C-terminus: V_(H)H-V_(H)-C_(H)1-C_(H)2-C_(H)3; and (b) a secondpolypeptide comprising from N-terminus to C-terminus: VL-CL, whereinV_(H) and V_(L) forms an antigen binding site that specifically bindsAng2, and wherein V_(H)H specifically binds VEGF. In some embodiments,the V_(H) and V_(L) domains are derived from LC10. In some embodiments,the V_(H) and V_(H)H domains are fused to each other via a peptidelinker, such as a peptide linker comprising the amino acid sequence ofSEQ ID NO: 1, 8 or 13. In some embodiments, the C_(H)2 and C_(H)3domains are derived from an IgG1 Fc. In some embodiments, the BABP hasthe structure as shown in FIG. 9 .

In some embodiments, there is provided a bispecific antigen bindingprotein comprising: (a) a first polypeptide comprising from N-terminusto C-terminus: V_(H)—C_(H)1-C_(H)2-C_(H)3; and (b) a second polypeptidecomprising from N-terminus to C-terminus: VL-CL-V_(H)H, wherein V_(H)and V_(L) forms an antigen binding site that specifically binds Ang2,and wherein V_(H)H specifically binds VEGF. In some embodiments, theV_(H) and V_(L) domains are derived from LC10. In some embodiments, theC_(L) and V_(H)H domains are fused to each other via a peptide linker,such as a peptide linker comprising the amino acid sequence of SEQ IDNO: 1, 8 or 13. In some embodiments, the C_(H)2 and C_(H)3 domains arederived from an IgG1 Fc. In some embodiments, the BABP has the structureas shown in FIG. 11 .

In some embodiments, there is provided a bispecific antigen bindingprotein comprising: (a) a first polypeptide comprising from N-terminusto C-terminus: V_(H)-C_(H)1-C_(H)2-C_(H)3; and (b) a second polypeptidecomprising from N-terminus to C-terminus: V_(H)H-VL-CL, wherein V_(H)and V_(L) forms an antigen binding site that specifically binds Ang2,and wherein V_(H)H specifically binds VEGF. In some embodiments, theV_(H) and V_(L) domains are derived from LC10. In some embodiments, theV_(L) and V_(H)H domains are fused to each other via a peptide linker,such as a peptide linker comprising the amino acid sequence of SEQ IDNO: 1, 8 or 13. In some embodiments, the C_(H)2 and C_(H)3 domains arederived from an IgG1 Fc. In some embodiments, the BABP has the structureas shown in FIG. 13 .

In some embodiments, there is provided a multispecific (such asbispecific) antigen binding protein comprising: (a) a first antigenbinding portion comprising a full-length antibody (such as adalimumab)consisting of two heavy chains and two light chains, wherein thefull-length antibody specifically binds TNF-α; and (b) a second antigenbinding portion comprising an sdAb that specifically binds IL-1β,wherein the first antigen binding portion and the second antigen bindingportion are fused to each other. In some embodiments, the sdAb is acamelid, humanized, or human sdAb. In some embodiments, the secondantigen binding portion is fused to the first antigen binding portion atthe N-terminus of one or each of the two heavy chains, the N-terminus ofone or each of the two light chains, the N-terminus of the Fc region,the C-terminus of one or each of the two heavy chains, or the C-terminusof one or each of the two light chains. In some embodiments, the secondantigen binding portion is fused to the first antigen binding portionchemically. In some embodiments, the second antigen binding portion isfused to the first antigen binding portion via a peptide bond or apeptide linker. In some embodiments, the peptide linker is no more thanabout 30 (such as no more than about any one of 25, 20, or 15) aminoacids long. In some embodiments, the peptide linker comprises the aminoacid sequence of SEQ ID NO: 1, 8 or 13. In some embodiments, the firstantigen binding fragment comprises an Fc region, such as an IgG1 Fc.

In some embodiments, there is provided a multispecific (such asbispecific) antigen binding protein comprising: (a) a first antigenbinding portion comprising a full-length antibody (such as mepolizumab)consisting of two heavy chains and two light chains, wherein thefull-length antibody specifically binds IL-5; and (b) a second antigenbinding portion comprising an sdAb that specifically binds eotaxin-1,wherein the first antigen binding portion and the second antigen bindingportion are fused to each other. In some embodiments, the sdAb is acamelid, humanized, or human sdAb. In some embodiments, the secondantigen binding portion is fused to the first antigen binding portion atthe N-terminus of one or each of the two heavy chains, the N-terminus ofone or each of the two light chains, the N-terminus of the Fc region,the C-terminus of one or each of the two heavy chains, or the C-terminusof one or each of the two light chains. In some embodiments, the secondantigen binding portion is fused to the first antigen binding portionchemically. In some embodiments, the second antigen binding portion isfused to the first antigen binding portion via a peptide bond or apeptide linker. In some embodiments, the peptide linker is no more thanabout 30 (such as no more than about any one of 25, 20, or 15) aminoacids long. In some embodiments, the peptide linker comprises the aminoacid sequence of SEQ ID NO: 1, 8 or 13. In some embodiments, the firstantigen binding fragment comprises an Fc region, such as an IgG1 Fc.

Properties of the MABPs

The MABPs described herein are amenable for manufacture and developmentas a biologic drug. In some embodiments, the MABP can be recombinantlyproduced at high expression levels. In some embodiments, the MABP can berecombinantly produced at a level sufficient for industrial production.In some embodiments, the MABP can be expressed transiently in mammaliancells. In some embodiments, the expression level of the MABP inmammalian cell culture is comparable to that of the parent 4-chainantibodies, such as no less than about any one of 50%, 60%, 70%, 80%,90%, 95%, or 100% solubility as the parent 4-chain antibody. As usedherein, the “parent 4-chain antibody” refers to an antibody, such as afull-length 4-chain antibody, comprising the VH and the VL of the firstantigen binding portion. In some embodiments, the expression level ofthe MABP in mammalian cell culture is higher than that of the parent4-chain antibodies. In some embodiments, the expression level of theMABP in mammalian cell culture (e.g., CHO cells) is at least about anyone of 10 mg/L, 15 mg/L, 20 mg/L, 30 mg/L, 40 mg/L, 50 mg/L, 60 mg/L, 70mg/L, 80 mg/L, 90 mg/L, 100 mg/L, 110 mg/L, 120 mg/L, 150 mg/L orhigher. Expression levels of the MABP in a cell culture can bedetermined using known methods in the art, such as by SDS-PAGE analysis,or analysis using a High-Performance Liquid Chromatography (HPLC) orFast Protein Liquid Chromatography (FPLC).

In some embodiments, the MABP produced by recombinant expression can bepurified to homogeneity or substantial homogeneity by a size exclusionchromatography. In some embodiments, the percentage of mono-dispersivemolecule (e.g., as a monomeric MABP molecule, such as a dimeric proteinconsisting of 4 polypeptide chains) in the purified MABP, e.g., asdetermined by chromatography, is at least about any one of 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or higher. The homogeneityof the MABP in a composition can be determined using known methods inthe art, such as by SDS-PAGE analysis, dynamic light scattering (DLS),or analysis using an HPLC or FPLC. In some embodiments, the yield of theMABP from the purification is at least about any one of 50%, 60%, 70%,80%, 90% or higher. In some embodiments, the yield of the MABP from thepurification is about 70% to about 95%.

The MABPs described herein further has various biophysical propertiesthat are amenable for use as a biologic drug, including, for example,high solubility, high long-term stability, and thermal stability.Stability of the MABP can be determined using known methods in the art,including Dynamic light scattering (DSL), which profiles differentpopulations of a molecule in soluble based on their particle sizes. Insome embodiments, at least about 90%, 91%, 92%, 93%, 94%, 95% or higherof the MABP in a composition is a non-aggregated conformation, i.e., assingle, monomeric MABP molecules, e.g., a dimeric protein consisting of4 polypeptide chains. In some embodiments, the level of aggregation,i.e., association of multiple MABP molecules as a complex, in acomposition is no more than about any one of 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10% or higher. In some embodiments, the time to form at leastabout 5% aggregation of the MABP in a composition is at least about anyone of 1 day, 3 days, 7 days, 2 weeks, 3 weeks, 4 weeks or more at about4° C. In some embodiments, the time to form at least about 5%aggregation of the MABP in a composition is at least about any one of 1day, 3 days, 7 days, 2 weeks, 3 weeks, 4 weeks or more at about roomtemperature, e.g., 25° C. In some embodiments, the time to form at leastabout 10% aggregation of the MABP in a composition is at least about anyone of 1 day, 2 days, 3 days, 4 days, 6 days, 7 days, 10 days, 2 weeksor more at physiological temperature, e.g., about 37° C.

In some embodiments, the MABP has comparable solubility, such as no lessthan about any one of 50%, 60%, 70%, 80%, 90%, 95%, or 100% solubilityas the parent 4-chain antibody or the sdAbs. In some embodiments, theMABP has higher solubility than the parent 4-chain antibodies or thesdAbs. In some embodiments, the MABP is soluble at a concentration of atleast about any one of 50 mg/mL, 75 mg/mL, 100 mg/mL, 125 mg/mL, 150mg/mL, 175 mg/mL, 200 mg/mL, 225 mg/mL, 250 mg/mL, 300 mg/mL or higher,for example, in a PBS buffer at pH 7.2. The solubility of the MABPs canbe measured using any known methods in the art, including concentrationusing a centrifugation filter followed by protein quantification, orpassing the MABP over an IgG-coupled cross-interaction chromatography(CIC) column. In some embodiments, the retention factor k′ of the MABPon a cross-interaction chromatography (CIC) column is no more than aboutany one of 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02,0.01 or less.

In some embodiments, the MABP has comparable thermal stability as theparent 4-chain antibody or antigen-binding fragment thereof. In someembodiments, the MABP has higher thermal stability than the parent4-chain antibodies or antigen-binding fragment thereof. Thermalstability can be measured using known methods in the art, includingCapillary Differential Scanning calorimetry (DSC) and DLS coupled togradual heating. In some embodiments, the MABP has an aggregation onsettemperature (T_(agg)) of at least about 65° C., such as at least aboutany one of 66° C., 67° C., 68° C., 69° C., 70° C., 71° C., 72° C., 73°C., 74° C., 75° C. or higher. In some embodiments, the MABP has anaggregation onset temperature (T_(agg)) of about 65° C. to about 75° C.In some embodiments, the MABP has an unfolding midpoint temperature(T_(m)) of at least about 65° C., such as at least about 66° C., 67° C.,68° C., 69° C., 70° C., 71° C., 72° C., 73° C., 74° C., 75° C. orhigher. In some embodiments, the MABP has an unfolding midpointtemperature (T_(m)) of about 65° C. to about 75° C.

In some embodiments, the MABP has a high long-term stability. In someembodiments, the MABP is stable for at least about any one of 1 day, 3days, 7 days, 2 weeks, 3 week, 4 weeks or more at about 4° C. In someembodiments, the MABP has a high long-term stability at an elevatedtemperature. In some embodiments, the MABP is stable for at least aboutany one of 1 day, 3 days, 7 days, 2 weeks, 3 week, 4 weeks or more atroom temperature, such as about 25° C. or higher. In some embodiments,the MABP is stable for at least about any one of 1 day, 2 days, 3 days,4 days, 6 days, 7 days, 10 days, 2 weeks or more at physiologicaltemperature, such as about 37° C. or higher. In some embodiments, thestability of the MABP is tested in an accelerated stability assessmentprogram, for example, at about any one of 40° C., 50° C., 60° C., 70° C.or higher do derive the stability of the MABP at a lower temperature. Insome embodiments, the MABP has a high long-term stability at a highconcentration, such as at least about any one of 50 mg/mL, 100 mg/mL,150 mg/mL, 200 mg/mL or higher. As used herein, a “stable” compositionis substantially free (such as less than about any of 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1% or less) of precipitation and/or aggregation.Precipitation can be detected by optical spectroscopy. Aggregation canbe detected by e.g., DLS.

In some embodiments, the MABP has high stability over freeze-thawcycles. In some embodiments, a composition comprising the MABP can befreeze-thawed for at least about any one of 3, 4, 5, 6, 7, 8, 9, 10times or more without losing structural integrity (e.g., formingaggregates) and/or activity of the MABP. In some embodiments, thecomposition comprising the MABP can be freeze-thawed at highconcentration, such as at least about any one of 50 mg/mL, 100 mg/mL,150 mg/mL, 200 mg/mL or higher.

Further provided are fragments derived from any one of the multispecificantigen binding proteins described herein, for example, Fab-likedomains.

III. Pharmaceutical Compositions

Further provided by the present application are pharmaceuticalcompositions comprising any one of the MABPs and a pharmaceuticallyacceptable carrier. Pharmaceutical compositions can be prepared bymixing a MABP having the desired degree of purity with optionalpharmaceutically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of lyophilized formulations or aqueous solutions.

In some embodiments, the pharmaceutical composition has a highconcentration of the MABP. In some embodiments, the concentration ofMABP in the pharmaceutical composition is at least about any one of 50mg/mL, 75 mg/mL, 100 mg/mL, 125 mg/mL, 150 mg/mL, 175 mg/mL, 200 mg/mL,225 mg/mL, 250 mg/mL, 300 mg/mL or higher. In some embodiments, thepharmaceutical composition has high thermal stability and long-termstability. In some embodiments, the pharmaceutical composition can bestored at about room temperature (e.g., about 25° C.) for at least aboutany one of 1 day, 3 days, 7 days, 2 weeks, 3 weeks, 4 weeks or more. Insome embodiments, the pharmaceutical composition can be stored at aphysiological temperature (e.g., about 37° C.) for at least about anyone of 1 day, 2 days, 3 days, 4 days, 5 days, 7 days, 10 days, 2 weeksor longer. In some embodiments, the pharmaceutical composition can befreeze-thawed for at least about any one of 3, 4, 5, 6, 7, 8, 9, 10times or more without losing structural integrity (e.g., formingaggregates) and/or activity of the MABP. In some embodiments, the shelflife of the pharmaceutical composition is at least about any one of 1weeks, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, or longer.

Acceptable carriers, excipients, or stabilizers are nontoxic torecipients at the dosages and concentrations employed, and includebuffers, antioxidants including ascorbic acid, methionine, Vitamin E,sodium metabisulfite; preservatives, isotonicifiers, stabilizers, metalcomplexes (e.g. Zn-protein complexes); chelating agents such as EDTAand/or non-ionic surfactants.

Buffers are used to control the pH in a range which optimizes thetherapeutic effectiveness, especially if stability is pH dependent.Buffers are preferably present at concentrations ranging from about 50mM to about 250 mM. Suitable buffering agents for use in the presentapplication include both organic and inorganic acids and salts thereof.For example, citrate, phosphate, succinate, tartrate, fumarate,gluconate, oxalate, lactate, acetate. Additionally, buffers may comprisehistidine and trimethylamine salts such as Tris.

Preservatives are added to retard microbial growth, and are typicallypresent in a range from 0.2%-1.0% (w/v). Suitable preservatives for usein the present application include octadecyldimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium halides (e.g., chloride,bromide, iodide), benzethonium chloride; thimerosal, phenol, butyl orbenzyl alcohol; alkyl parabens such as methyl or propyl paraben;catechol; resorcinol; cyclohexanol, 3-pentanol, and m-cresol.

Tonicity agents, sometimes known as “stabilizers” are present to adjustor maintain the tonicity of liquid in a composition. When used withlarge, charged biomolecules such as proteins and antibodies, they areoften termed “stabilizers” because they can interact with the chargedgroups of the amino acid side chains, thereby lessening the potentialfor inter and intra-molecular interactions. Tonicity agents can bepresent in any amount between 0.1% to 25% by weight, preferably 1 to 5%,taking into account the relative amounts of the other ingredients.Preferred tonicity agents include polyhydric sugar alcohols, preferablytrihydric or higher sugar alcohols, such as glycerin, erythritol,arabitol, xylitol, sorbitol and mannitol.

Additional excipients include agents which can serve as one or more ofthe following: (1) bulking agents, (2) solubility enhancers, (3)stabilizers and (4) and agents preventing denaturation or adherence tothe container wall. Such excipients include: polyhydric sugar alcohols(enumerated above); amino acids such as alanine, glycine, glutamine,asparagine, histidine, arginine, lysine, ornithine, leucine,2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugaralcohols such as sucrose, lactose, lactitol, trehalose, stachyose,mannose, sorbose, xylose, ribose, ribitol, myoinisitose, myoinisitol,galactose, galactitol, glycerol, cyclitols (e.g., inositol),polyethylene glycol; sulfur containing reducing agents, such as urea,glutathione, thioctic acid, sodium thioglycolate, thioglycerol,α-monothioglycerol and sodium thio sulfate; low molecular weightproteins such as human serum albumin, bovine serum albumin, gelatin orother immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; monosaccharides (e.g., xylose, mannose, fructose,glucose; disaccharides (e.g., lactose, maltose, sucrose); trisaccharidessuch as raffinose; and polysaccharides such as dextrin or dextran.

Non-ionic surfactants or detergents (also known as “wetting agents”) arepresent to help solubilize the therapeutic agent as well as to protectthe therapeutic protein against agitation-induced aggregation, whichalso permits the formulation to be exposed to shear surface stresswithout causing denaturation of the active therapeutic protein orantibody. Non-ionic surfactants are present in a range of about 0.05mg/ml to about 1.0 mg/ml, preferably about 0.07 mg/ml to about 0.2mg/ml.

Suitable non-ionic surfactants include polysorbates (20, 40, 60, 65, 80,etc.), polyoxamers (184, 188, etc.), PLURONIC® polyols, TRITON®,polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.),lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenatedcastor oil 10, 50 and 60, glycerol monostearate, sucrose fatty acidester, methyl celluose and carboxymethyl cellulose. Anionic detergentsthat can be used include sodium lauryl sulfate, dioctyle sodiumsulfosuccinate and dioctyl sodium sulfonate. Cationic detergents includebenzalkonium chloride or benzethonium chloride.

In order for the pharmaceutical compositions to be used for in vivoadministration, they must be sterile. The pharmaceutical composition maybe rendered sterile by filtration through sterile filtration membranes.The pharmaceutical compositions herein generally are placed into acontainer having a sterile access port, for example, an intravenoussolution bag or vial having a stopper pierceable by a hypodermicinjection needle.

The route of administration is in accordance with known and acceptedmethods, such as by single or multiple bolus or infusion over a longperiod of time in a suitable manner, e.g., injection or infusion bysubcutaneous, intravenous, intraperitoneal, intramuscular,intraarterial, intralesional or intraarticular routes, topicaladministration, inhalation or by sustained release or extended-releasemeans.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semi-permeable matrices of solidhydrophobic polymers containing the antagonist, which matrices are inthe form of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and.ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.

The pharmaceutical compositions herein may also contain more than oneactive compound as necessary for the particular indication beingtreated, preferably those with complementary activities that do notadversely affect each other. Alternatively, or in addition, thecomposition may comprise a cytotoxic agent, chemotherapeutic agent,cytokine, immunosuppressive agent, or growth inhibitory agent. Suchmolecules are suitably present in combination in amounts that areeffective for the purpose intended.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coascervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 18th edition.

An exemplary pharmaceutical formulation of the MABP is a liquidformulation comprising sodium citrate, sodium chloride, mannitol,diethylenetriaminepentacetic acid (pentetic acid), and polysorbate 80(Tween 80), at pH 6.0. In some embodiments, the MABP is formulated in aliquid formulation comprising 4% Sucrose, 50 mM Histidine, 50 mMArginine, at pH 6.0.

IV. Methods of Use

The multispecific antigen binding proteins described herein, and thecompositions (such as pharmaceutical compositions) thereof are usefulfor a variety of applications, such as in diagnosis, molecular assays,and therapy.

In some embodiments, there is a method of treating a disease or acondition in an individual in need thereof, comprising administering aneffective amount of a pharmaceutical composition comprising amultispecific (such as bispecific) antigen binding protein and apharmaceutically acceptable carrier, wherein the MABP comprises (a) afirst antigen binding portion comprising a heavy chain variable domain(V_(H)) and a light chain variable domain (V_(L)), wherein the V_(H) andV_(L) together form an antigen-binding site that specifically binds afirst epitope, and (b) a second antigen binding portion comprising ansdAb that specifically binds a second epitope, wherein the first antigenbinding portion and the second antigen binding portion are fused to eachother. In some embodiments, the sdAb is a camelid, humanized, or humansdAb. In some embodiments, the first antigen binding portion comprises aheavy chain comprising the V_(H) and a light chain comprising the V_(L).In some embodiments, the second antigen binding portion is fused to thefirst antigen binding portion at the N-terminus of the heavy chain, theN-terminus of the light chain, the N-terminus of the Fc region, theC-terminus of the heavy chain, or the C-terminus of the light chain. Insome embodiments, the first antigen binding portion comprises afull-length 4-chain antibody. In some embodiments, the second antigenbinding portion is fused to the first antigen binding portionchemically. In some embodiments, the second antigen binding portion isfused to the first antigen binding portion via a peptide bond or apeptide linker. In some embodiments, the peptide linker is no more thanabout 30 (such as no more than about any one of 25, 20, or 15) aminoacids long. In some embodiments, the first antigen binding fragmentcomprises an Fc region, such as an IgG1 Fc or IgG4 Fc.

Methods of Treating a Cancer

In some embodiments, there is provided a method of treating a cancer inan individual in need thereof, comprising administering an effectiveamount of a pharmaceutical composition comprising a multispecific (suchas bispecific) antigen binding protein and a pharmaceutically acceptablecarrier, wherein the MABP comprises: (a) a first antigen binding portioncomprising a heavy chain variable domain (V_(H)) and a light chainvariable domain (V_(L)), wherein the V_(H) and V_(L) together form anantigen-binding site that specifically binds a first epitope, and (b) asecond antigen binding portion comprising an sdAb that specificallybinds a second epitope, wherein the first antigen binding portion andthe second antigen binding portion are fused to each other. In someembodiments, the sdAb is a camelid, humanized, or human sdAb. In someembodiments, the cancer is selected from the group consisting of breastcancer, renal cancer, melanoma, lung cancer, glioblastoma, head and neckcancer, prostate cancer, ovarian carcinoma, bladder carcinoma, andlymphoma. In some embodiments, the first antigen binding portioncomprises a heavy chain comprising the V_(H) and a light chaincomprising the V_(L). In some embodiments, the second antigen bindingportion is fused to the first antigen binding portion at the N-terminusof the heavy chain, the N-terminus of the light chain, the N-terminus ofthe Fc region, the C-terminus of the heavy chain, or the C-terminus ofthe light chain. In some embodiments, the first antigen binding portioncomprises a full-length 4-chain antibody. In some embodiments, thesecond antigen binding portion is fused to the first antigen bindingportion chemically. In some embodiments, the second antigen bindingportion is fused to the first antigen binding portion via a peptide bondor a peptide linker. In some embodiments, the peptide linker is no morethan about 30 (such as no more than about any one of 25, 20, or 15)amino acids long. In some embodiments, the first antigen bindingfragment comprises an Fc region, such as an IgG1 Fc or IgG4 Fc.

In some embodiments, there is provided a method of treating a cancer inan individual in need thereof, comprising administering an effectiveamount of a pharmaceutical composition comprising a multispecific (suchas bispecific) antigen binding protein and a pharmaceutically acceptablecarrier, wherein the MABP comprises: (a) a first antigen binding portioncomprising a heavy chain variable domain (V_(H)) and a light chainvariable domain (V_(L)), wherein the V_(H) and V_(L) together form anantigen-binding site that specifically binds a first immune checkpointmolecule, and (b) a second antigen binding portion comprising an sdAb(e.g., a V_(H)H) that specifically binds a second immune checkpointmolecule, wherein the first antigen binding portion and the secondantigen binding portion are fused to each other. In some embodiments,the sdAb is a camelid, humanized, or human sdAb. In some embodiments,the first immune checkpoint molecule and/or the second immune checkpointmolecule is selected from the group consisting of PD-1, PD-L1, PD-L2,CTLA-4, B7-H3, TIM-3, LAG-3, VISTA, ICOS, 4-1BB, OX40, GITR, and CD40.In some embodiments, the cancer is selected from the group consisting ofbreast cancer, renal cancer, melanoma, lung cancer, glioblastoma, headand neck cancer, prostate cancer, ovarian carcinoma, bladder carcinoma,and lymphoma. In some embodiments, the first antigen binding portioncomprises a heavy chain comprising the V_(H) and a light chaincomprising the V_(L). In some embodiments, the second antigen bindingportion is fused to the first antigen binding portion at the N-terminusof the heavy chain, the N-terminus of the light chain, the N-terminus ofthe Fc region, the C-terminus of the heavy chain, or the C-terminus ofthe light chain. In some embodiments, the first antigen binding portioncomprises a full-length 4-chain antibody. In some embodiments, thesecond antigen binding portion is fused to the first antigen bindingportion chemically. In some embodiments, the second antigen bindingportion is fused to the first antigen binding portion via a peptide bondor a peptide linker. In some embodiments, the peptide linker is no morethan about 30 (such as no more than about any one of 25, 20, or 15)amino acids long. In some embodiments, the first antigen bindingfragment comprises an Fc region, such as an IgG4 Fc.

In some embodiments, there is provided a method of treating a cancer inan individual in need thereof, comprising administering an effectiveamount of a pharmaceutical composition comprising a multispecific (suchas bispecific) antigen binding protein and a pharmaceutically acceptablecarrier, wherein the MABP comprises: (a) a first antigen binding portioncomprising a full-length antibody (such as pembrolizumab or nivolumab)consisting of two heavy chains and two light chains, wherein thefull-length antibody specifically binds PD-1; and (b) a second antigenbinding portion comprising an sdAb (e.g., a V_(H)H) that specificallybinds CTLA-4, wherein the first antigen binding portion and the secondantigen binding portion are fused to each other. In some embodiments,the sdAb is a camelid, humanized, or human sdAb. In some embodiments,the sdAb binds CTLA-4 with a high affinity. In some embodiments, thesdAb binds CTLA-4 with a medium affinity. In some embodiments, the sdAbbinds CTLA-4 with a low affinity. In some embodiments, the cancer isselected from the group consisting of breast cancer, renal cancer,melanoma, lung cancer, glioblastoma, head and neck cancer, prostatecancer, ovarian carcinoma, bladder carcinoma, and lymphoma. In someembodiments, the second antigen binding portion is fused to the firstantigen binding portion at the N-terminus of one or each of the twoheavy chains, the N-terminus of one or each of the two light chains, theN-terminus of the Fc region, the C-terminus of one or each of the twoheavy chains, or the C-terminus of one or each of the two light chains.In some embodiments, the second antigen binding portion is fused to thefirst antigen binding portion chemically. In some embodiments, thesecond antigen binding portion is fused to the first antigen bindingportion via a peptide bond or a peptide linker. In some embodiments, thepeptide linker is no more than about 30 (such as no more than about anyone of 25, 20, or 15) amino acids long. In some embodiments, the firstantigen binding fragment comprises an Fc region, such as an IgG4 Fc.

In some embodiments, there is provided a method of treating a cancer inan individual in need thereof, comprising administering an effectiveamount of a pharmaceutical composition comprising a multispecific (suchas bispecific) antigen binding protein and a pharmaceutically acceptablecarrier, wherein the MABP comprises: (a) a first antigen binding portioncomprising a full-length antibody (such as pembrolizumab or nivolumab)consisting of two heavy chains and two light chains, wherein thefull-length antibody specifically binds PD-1; and (b) a second antigenbinding portion comprising an sdAb that specifically binds TIM-3,wherein the first antigen binding portion and the second antigen bindingportion are fused to each other. In some embodiments, the sdAb is acamelid, humanized, or human sdAb. In some embodiments, the cancer isselected from the group consisting of breast cancer, renal cancer,melanoma, lung cancer, glioblastoma, head and neck cancer, prostatecancer, ovarian carcinoma, bladder carcinoma, and lymphoma. In someembodiments, the second antigen binding portion is fused to the firstantigen binding portion at the N-terminus of one or each of the twoheavy chains, the N-terminus of one or each of the two light chains, theN-terminus of the Fc region, the C-terminus of one or each of the twoheavy chains, or the C-terminus of one or each of the two light chains.In some embodiments, the second antigen binding portion is fused to thefirst antigen binding portion chemically. In some embodiments, thesecond antigen binding portion is fused to the first antigen bindingportion via a peptide bond or a peptide linker. In some embodiments, thepeptide linker is no more than about 30 (such as no more than about anyone of 25, 20, or 15) amino acids long. In some embodiments, the firstantigen binding fragment comprises an Fc region, such as an IgG4 Fc.

In some embodiments, there is provided a method of treating a cancer inan individual in need thereof, comprising administering an effectiveamount of a pharmaceutical composition comprising a multispecific (suchas bispecific) antigen binding protein and a pharmaceutically acceptablecarrier, wherein the MABP comprises: (a) a first antigen binding portioncomprising a full-length antibody (such as pembrolizumab or nivolumab)consisting of two heavy chains and two light chains, wherein thefull-length antibody specifically binds PD-1; and (b) a second antigenbinding portion comprising an sdAb that specifically binds LAG-3,wherein the first antigen binding portion and the second antigen bindingportion are fused to each other. In some embodiments, the sdAb is acamelid, humanized, or human sdAb. In some embodiments, the cancer isselected from the group consisting of breast cancer, renal cancer,melanoma, lung cancer, glioblastoma, head and neck cancer, prostatecancer, ovarian carcinoma, bladder carcinoma, and lymphoma. In someembodiments, the second antigen binding portion is fused to the firstantigen binding portion at the N-terminus of one or each of the twoheavy chains, the N-terminus of one or each of the two light chains, theN-terminus of the Fc region, the C-terminus of one or each of the twoheavy chains, or the C-terminus of one or each of the two light chains.In some embodiments, the second antigen binding portion is fused to thefirst antigen binding portion chemically. In some embodiments, thesecond antigen binding portion is fused to the first antigen bindingportion via a peptide bond or a peptide linker. In some embodiments, thepeptide linker is no more than about 30 (such as no more than about anyone of 25, 20, or 15) amino acids long. In some embodiments, the firstantigen binding fragment comprises an Fc region, such as an IgG4 Fc.

In some embodiments, there is provided a method of treating a cancer inan individual in need thereof, comprising administering an effectiveamount of a pharmaceutical composition comprising a multispecific (suchas bispecific) antigen binding protein and a pharmaceutically acceptablecarrier, wherein the MABP comprises: (a) a first antigen binding portioncomprising a full-length antibody (such as pembrolizumab or nivolumab)consisting of two heavy chains and two light chains, wherein thefull-length antibody specifically binds PD-1; and (b) a second antigenbinding portion comprising an sdAb that specifically binds VISTA,wherein the first antigen binding portion and the second antigen bindingportion are fused to each other. In some embodiments, the sdAb is acamelid, humanized, or human sdAb. In some embodiments, the cancer isselected from the group consisting of breast cancer, renal cancer,melanoma, lung cancer, glioblastoma, head and neck cancer, prostatecancer, ovarian carcinoma, bladder carcinoma, and lymphoma. In someembodiments, the second antigen binding portion is fused to the firstantigen binding portion at the N-terminus of one or each of the twoheavy chains, the N-terminus of one or each of the two light chains, theN-terminus of the Fc region, the C-terminus of one or each of the twoheavy chains, or the C-terminus of one or each of the two light chains.In some embodiments, the second antigen binding portion is fused to thefirst antigen binding portion chemically. In some embodiments, thesecond antigen binding portion is fused to the first antigen bindingportion via a peptide bond or a peptide linker. In some embodiments, thepeptide linker is no more than about 30 (such as no more than about anyone of 25, 20, or 15) amino acids long. In some embodiments, the firstantigen binding fragment comprises an Fc region, such as an IgG4 Fc.

In some embodiments, there is provided a method of treating a cancer inan individual in need thereof, comprising administering an effectiveamount of a pharmaceutical composition comprising a multispecific (suchas bispecific) antigen binding protein and a pharmaceutically acceptablecarrier, wherein the MABP comprises: (a) a first antigen binding portioncomprising pembrolizumab consisting of two heavy chains and two lightchains; and (b) a second antigen binding portion comprising ananti-CTLA-4 sdAb, wherein the first antigen binding portion and thesecond antigen binding portion are fused to each other. In someembodiments, the sdAb is a camelid, humanized, or human sdAb. In someembodiments, the cancer is selected from the group consisting of breastcancer, renal cancer, melanoma, lung cancer, glioblastoma, head and neckcancer, prostate cancer, ovarian carcinoma, bladder carcinoma, andlymphoma. In some embodiments, the second antigen binding portion isfused to the first antigen binding portion at the N-terminus of one oreach of the two heavy chains, the N-terminus of one or each of the twolight chains, the N-terminus of the Fc region, the C-terminus of one oreach of the two heavy chains, or the C-terminus of one or each of thetwo light chains. In some embodiments, the second antigen bindingportion is fused to the first antigen binding portion chemically. Insome embodiments, the second antigen binding portion is fused to thefirst antigen binding portion via a peptide bond or a peptide linker. Insome embodiments, the peptide linker is no more than about 30 (such asno more than about any one of 25, 20, or 15) amino acids long. In someembodiments, the peptide linker comprises the amino acid sequence of SEQID NO: 1, 8 or 13. In some embodiments, the first antigen bindingfragment comprises an Fc region, such as an IgG4 Fc.

In some embodiments, there is provided a method of treating a cancer inan individual in need thereof, comprising administering an effectiveamount of a pharmaceutical composition comprising a multispecific (suchas bispecific) antigen binding protein and a pharmaceutically acceptablecarrier, wherein the MABP comprises: (a) a first antigen binding portioncomprising a full-length antibody (such as durvalumab or atezolizumab)consisting of two heavy chains and two light chains, wherein thefull-length antibody specifically binds PD-L1; and (b) a second antigenbinding portion comprising an sdAb that specifically binds CTLA-4,wherein the first antigen binding portion and the second antigen bindingportion are fused to each other. In some embodiments, the sdAb is acamelid, humanized, or human sdAb. In some embodiments, the sdAb bindsCTLA-4 with a high affinity. In some embodiments, the sdAb binds CTLA-4with a medium affinity. In some embodiments, the sdAb binds CTLA-4 witha low affinity. In some embodiments, the cancer is selected from thegroup consisting of breast cancer, renal cancer, melanoma, lung cancer,glioblastoma, head and neck cancer, prostate cancer, ovarian carcinoma,bladder carcinoma, and lymphoma. In some embodiments, the second antigenbinding portion is fused to the first antigen binding portion at theN-terminus of one or each of the two heavy chains, the N-terminus of oneor each of the two light chains, the N-terminus of the Fc region, theC-terminus of one or each of the two heavy chains, or the C-terminus ofone or each of the two light chains. In some embodiments, the secondantigen binding portion is fused to the first antigen binding portionchemically. In some embodiments, the second antigen binding portion isfused to the first antigen binding portion via a peptide bond or apeptide linker. In some embodiments, the peptide linker is no more thanabout 30 (such as no more than about any one of 25, 20, or 15) aminoacids long. In some embodiments, the first antigen binding fragmentcomprises an Fc region, such as an IgG4 Fc.

In some embodiments, there is provided a method of treating a cancer inan individual in need thereof, comprising administering an effectiveamount of a pharmaceutical composition comprising a multispecific (suchas bispecific) antigen binding protein and a pharmaceutically acceptablecarrier, wherein the MABP comprises: (a) a first antigen binding portioncomprising a full-length antibody (such as durvalumab or atezolizumab)consisting of two heavy chains and two light chains, wherein thefull-length antibody specifically binds PD-L1; and (b) a second antigenbinding portion comprising an sdAb that specifically binds TIM-3,wherein the first antigen binding portion and the second antigen bindingportion are fused to each other. In some embodiments, the sdAb is acamelid, humanized, or human sdAb. In some embodiments, the cancer isselected from the group consisting of breast cancer, renal cancer,melanoma, lung cancer, glioblastoma, head and neck cancer, prostatecancer, ovarian carcinoma, bladder carcinoma, and lymphoma. In someembodiments, the second antigen binding portion is fused to the firstantigen binding portion at the N-terminus of one or each of the twoheavy chains, the N-terminus of one or each of the two light chains, theN-terminus of the Fc region, the C-terminus of one or each of the twoheavy chains, or the C-terminus of one or each of the two light chains.In some embodiments, the second antigen binding portion is fused to thefirst antigen binding portion chemically. In some embodiments, thesecond antigen binding portion is fused to the first antigen bindingportion via a peptide bond or a peptide linker. In some embodiments, thepeptide linker is no more than about 30 (such as no more than about anyone of 25, 20, or 15) amino acids long. In some embodiments, the firstantigen binding fragment comprises an Fc region, such as an IgG4 Fc.

In some embodiments, there is provided a method of treating a cancer inan individual in need thereof, comprising administering an effectiveamount of a pharmaceutical composition comprising a multispecific (suchas bispecific) antigen binding protein and a pharmaceutically acceptablecarrier, wherein the MABP comprises: (a) a first antigen binding portioncomprising a full-length antibody (such as durvalumab or atezolizumab)consisting of two heavy chains and two light chains, wherein thefull-length antibody specifically binds PD-L1; and (b) a second antigenbinding portion comprising an sdAb that specifically binds LAG-3,wherein the first antigen binding portion and the second antigen bindingportion are fused to each other. In some embodiments, the sdAb is acamelid, humanized, or human sdAb. In some embodiments, the cancer isselected from the group consisting of breast cancer, renal cancer,melanoma, lung cancer, glioblastoma, head and neck cancer, prostatecancer, ovarian carcinoma, bladder carcinoma, and lymphoma. In someembodiments, the second antigen binding portion is fused to the firstantigen binding portion at the N-terminus of one or each of the twoheavy chains, the N-terminus of one or each of the two light chains, theN-terminus of the Fc region, the C-terminus of one or each of the twoheavy chains, or the C-terminus of one or each of the two light chains.In some embodiments, the second antigen binding portion is fused to thefirst antigen binding portion chemically. In some embodiments, thesecond antigen binding portion is fused to the first antigen bindingportion via a peptide bond or a peptide linker. In some embodiments, thepeptide linker is no more than about 30 (such as no more than about anyone of 25, 20, or 15) amino acids long. In some embodiments, the firstantigen binding fragment comprises an Fc region, such as an IgG4 Fc.

In some embodiments, there is provided a method of treating a cancer inan individual in need thereof, comprising administering an effectiveamount of a pharmaceutical composition comprising a multispecific (suchas bispecific) antigen binding protein and a pharmaceutically acceptablecarrier, wherein the MABP comprises: (a) a first antigen binding portioncomprising a full-length antibody (such as durvalumab or atezolizumab)consisting of two heavy chains and two light chains, wherein thefull-length antibody specifically binds PD-L1; and (b) a second antigenbinding portion comprising an sdAb that specifically binds VISTA,wherein the first antigen binding portion and the second antigen bindingportion are fused to each other. In some embodiments, the sdAb is acamelid, humanized, or human sdAb. In some embodiments, the cancer isselected from the group consisting of breast cancer, renal cancer,melanoma, lung cancer, glioblastoma, head and neck cancer, prostatecancer, ovarian carcinoma, bladder carcinoma, and lymphoma. In someembodiments, the second antigen binding portion is fused to the firstantigen binding portion at the N-terminus of one or each of the twoheavy chains, the N-terminus of one or each of the two light chains, theN-terminus of the Fc region, the C-terminus of one or each of the twoheavy chains, or the C-terminus of one or each of the two light chains.In some embodiments, the second antigen binding portion is fused to thefirst antigen binding portion chemically. In some embodiments, thesecond antigen binding portion is fused to the first antigen bindingportion via a peptide bond or a peptide linker. In some embodiments, thepeptide linker is no more than about 30 (such as no more than about anyone of 25, 20, or 15) amino acids long. In some embodiments, the firstantigen binding fragment comprises an Fc region, such as an IgG4 Fc.

In some embodiments, there is provided a method of treating a cancer inan individual in need thereof, comprising administering an effectiveamount of a pharmaceutical composition comprising a multispecific (suchas bispecific) antigen binding protein and a pharmaceutically acceptablecarrier, wherein the MABP comprises: (a) a first antigen binding portioncomprising a heavy chain variable domain (V_(H)) and a light chainvariable domain (V_(L)), wherein the V_(H) and V_(L) together form anantigen-binding site that specifically binds a first tumor antigen, and(b) a second antigen binding portion comprising an sdAb (e.g., a V_(H)H)that specifically binds a second tumor antigen, wherein the firstantigen binding portion and the second antigen binding portion are fusedto each other. In some embodiments, the sdAb is a camelid, humanized, orhuman sdAb. In some embodiments, the first tumor antigen and/or thesecond tumor antigen is selected from the group consisting of HER2,BRAF, EGFR, VEGFR2, CD20, RANKL, CD38, and CD52. In some embodiments,the first antigen binding portion comprises a full-length anti-HER-2monoclonal antibody (such as trastuzumab) or antigen binding fragmentthereof. In some embodiments, the cancer is selected from the groupconsisting of breast cancer, renal cancer, melanoma, lung cancer,glioblastoma, head and neck cancer, prostate cancer, ovarian carcinoma,bladder carcinoma, and lymphoma. In some embodiments, the second antigenbinding portion is fused to the first antigen binding portion at theN-terminus of one or each of the two heavy chains, the N-terminus of oneor each of the two light chains, the N-terminus of the Fc region, theC-terminus of one or each of the two heavy chains, or the C-terminus ofone or each of the two light chains. In some embodiments, the secondantigen binding portion is fused to the first antigen binding portionchemically. In some embodiments, the second antigen binding portion isfused to the first antigen binding portion via a peptide bond or apeptide linker. In some embodiments, the peptide linker is no more thanabout 30 (such as no more than about any one of 25, 20, or 15) aminoacids long. In some embodiments, the first antigen binding fragmentcomprises an Fc region, such as an IgG1 Fc.

In some embodiments, there is provided a method of treating a cancer inan individual in need thereof, comprising administering an effectiveamount of a pharmaceutical composition comprising a multispecific (suchas bispecific) antigen binding protein and a pharmaceutically acceptablecarrier, wherein the MABP comprises: (a) a first antigen binding portioncomprising a heavy chain variable domain (V_(H)) and a light chainvariable domain (V_(L)), wherein the V_(H) and V_(L) together form anantigen-binding site that specifically binds a tumor antigen, and (b) asecond antigen binding portion comprising an sdAb (e.g., a V_(H)H) thatspecifically binds a cell surface antigen on an immune effector cell(such as T cell), wherein the first antigen binding portion and thesecond antigen binding portion are fused to each other. In someembodiments, the sdAb is a camelid, humanized, or human sdAb. In someembodiments, the tumor antigen is selected from the group consisting ofHER2, BRAF, EGFR, VEGFR2, CD20, RANKL, CD38, and CD52. In someembodiments, the first antigen binding portion comprises a full-lengthanti-HER-2 monoclonal antibody (such as trastuzumab) or antigen bindingfragment thereof. In some embodiments, the cancer is selected from thegroup consisting of breast cancer, renal cancer, melanoma, lung cancer,glioblastoma, head and neck cancer, prostate cancer, ovarian carcinoma,bladder carcinoma, and lymphoma. In some embodiments, the second antigenbinding portion is fused to the first antigen binding portion at theN-terminus of one or each of the two heavy chains, the N-terminus of oneor each of the two light chains, the N-terminus of the Fc region, theC-terminus of one or each of the two heavy chains, or the C-terminus ofone or each of the two light chains. In some embodiments, the secondantigen binding portion is fused to the first antigen binding portionchemically. In some embodiments, the second antigen binding portion isfused to the first antigen binding portion via a peptide bond or apeptide linker. In some embodiments, the peptide linker is no more thanabout 30 (such as no more than about any one of 25, 20, or 15) aminoacids long. In some embodiments, the first antigen binding fragmentcomprises an Fc region, such as an IgG1 Fc.

In some embodiments, there is provided a method of treating a cancer inan individual in need thereof, comprising administering an effectiveamount of a pharmaceutical composition comprising a multispecific (suchas bispecific) antigen binding protein and a pharmaceutically acceptablecarrier, wherein the MABP comprises: (a) a first antigen binding portioncomprising a heavy chain variable domain (V_(H)) and a light chainvariable domain (V_(L)), wherein the V_(H) and V_(L) together form anantigen-binding site that specifically binds a first angiogenic factor(such as Ang-2), and (b) a second antigen binding portion comprising ansdAb (e.g., a V_(H)H) that specifically binds a second angiogenic factor(such as VEGF), wherein the first antigen binding portion and the secondantigen binding portion are fused to each other. In some embodiments,the sdAb is a camelid, humanized, or human sdAb. In some embodiments,the cancer is selected from the group consisting of breast cancer, renalcancer, melanoma, lung cancer, glioblastoma, head and neck cancer,prostate cancer, ovarian carcinoma, bladder carcinoma, and lymphoma. Insome embodiments, the second antigen binding portion is fused to thefirst antigen binding portion at the N-terminus of one or each of thetwo heavy chains, the N-terminus of one or each of the two light chains,the N-terminus of the Fc region, the C-terminus of one or each of thetwo heavy chains, or the C-terminus of one or each of the two lightchains. In some embodiments, the second antigen binding portion is fusedto the first antigen binding portion chemically. In some embodiments,the second antigen binding portion is fused to the first antigen bindingportion via a peptide bond or a peptide linker. In some embodiments, thepeptide linker is no more than about 30 (such as no more than about anyone of 25, 20, or 15) amino acids long. In some embodiments, the firstantigen binding fragment comprises an Fc region, such as an IgG1 Fc.

In some embodiments, there is provided a method of treating a cancer(such as breast cancer) in an individual in need thereof, comprisingadministering an effective amount of a pharmaceutical compositioncomprising a multispecific (such as bispecific) antigen binding proteinand a pharmaceutically acceptable carrier, wherein the MABP comprises:(a) a first antigen binding portion comprising a full-length antibody(such as trastuzumab) consisting of two heavy chains and two lightchains, wherein the full-length antibody specifically binds HER2receptor; and (b) a second antigen binding portion comprising an sdAbthat specifically binds CD3, wherein the first antigen binding portionand the second antigen binding portion are fused to each other. In someembodiments, the sdAb is a camelid, humanized, or human sdAb. In someembodiments, the second antigen binding portion is fused to the firstantigen binding portion at the N-terminus of one or each of the twoheavy chains, the N-terminus of one or each of the two light chains, theN-terminus of the Fc region, the C-terminus of one or each of the twoheavy chains, or the C-terminus of one or each of the two light chains.In some embodiments, the second antigen binding portion is fused to thefirst antigen binding portion chemically. In some embodiments, thesecond antigen binding portion is fused to the first antigen bindingportion via a peptide bond or a peptide linker. In some embodiments, thepeptide linker is no more than about 30 (such as no more than about anyone of 25, 20, or 15) amino acids long. In some embodiments, the firstantigen binding fragment comprises an Fc region, such as an IgG4 Fc.

The methods described herein are suitable for treating various cancers,including both solid cancer and liquid cancer. The methods areapplicable to cancers of all stages, including early stage, advancedstage and metastatic cancer. The methods described herein may be used asa first therapy, second therapy, third therapy, or combination therapywith other types of cancer therapies known in the art, such aschemotherapy, surgery, radiation, gene therapy, immunotherapy, bonemarrow transplantation, stem cell transplantation, targeted therapy,cryotherapy, ultrasound therapy, photodynamic therapy, radio-frequencyablation or the like, in an adjuvant setting or a neoadjuvant setting.

Methods of Treating Inflammatory or Autoimmune Disease

In some embodiments, there is provided a method of treating aninflammatory or autoimmune disease in an individual in need thereof,comprising administering an effective amount of a pharmaceuticalcomposition comprising a multispecific (such as bispecific) antigenbinding protein and a pharmaceutically acceptable carrier, wherein theMABP comprises: (a) a first antigen binding portion comprising a heavychain variable domain (V_(H)) and a light chain variable domain (V_(L)),wherein the V_(H) and V_(L) together form an antigen-binding site thatspecifically binds a first epitope, and (b) a second antigen bindingportion comprising an sdAb that specifically binds a second epitope,wherein the first antigen binding portion and the second antigen bindingportion are fused to each other. In some embodiments, the sdAb is acamelid, humanized, or human sdAb. In some embodiments, the inflammatoryor autoimmune disease is selected from the group consisting of arthritis(such as rheumatoid arthritis, juvenile idiopathic arthritis, psoriaticarthritis, ankylosing spondylitis, and arthritic ulcerative colitis),colitis, psoriasis, severe asthma, and moderate to severe Cronh'sdisease. In some embodiments, the second antigen binding portion isfused to the first antigen binding portion at the N-terminus of one oreach of the two heavy chains, the N-terminus of one or each of the twolight chains, the N-terminus of the Fc region, the C-terminus of one oreach of the two heavy chains, or the C-terminus of one or each of thetwo light chains. In some embodiments, the second antigen bindingportion is fused to the first antigen binding portion chemically. Insome embodiments, the second antigen binding portion is fused to thefirst antigen binding portion via a peptide bond or a peptide linker. Insome embodiments, the peptide linker is no more than about 30 (such asno more than about any one of 25, 20, or 15) amino acids long. In someembodiments, the first antigen binding fragment comprises an Fc region,such as an IgG1 Fc.

In some embodiments, there is provided a method of treating aninflammatory or autoimmune disease in an individual in need thereof,comprising administering an effective amount of a pharmaceuticalcomposition comprising a multispecific (such as bispecific) antigenbinding protein and a pharmaceutically acceptable carrier, wherein theMABP comprises: (a) a first antigen binding portion comprising a heavychain variable domain (V_(H)) and a light chain variable domain (V_(L)),wherein the V_(H) and V_(L) together form an antigen-binding site thatspecifically binds a first pro-inflammatory molecule, and (b) a secondantigen binding portion comprising an sdAb that specifically binds asecond pro-inflammatory molecule, wherein the first antigen bindingportion and the second antigen binding portion are fused to each other.In some embodiments, the sdAb is a camelid, humanized, or human sdAb. Insome embodiments, the inflammatory or autoimmune disease is selectedfrom the group consisting of arthritis (such as rheumatoid arthritis,juvenile idiopathic arthritis, psoriatic arthritis, ankylosingspondylitis, and arthritic ulcerative colitis), colitis, psoriasis,severe asthma, and moderate to severe Cronh's disease. In someembodiments, the first pro-inflammatory molecule and/or the secondpro-inflammatory molecule is selected from the group consisting ofIL-1β, TNF-α, IL-5, IL-6, IL-6R, and eotaxin-1. In some embodiments, thesecond antigen binding portion is fused to the first antigen bindingportion at the N-terminus of one or each of the two heavy chains, theN-terminus of one or each of the two light chains, the N-terminus of theFc region, the C-terminus of one or each of the two heavy chains, or theC-terminus of one or each of the two light chains. In some embodiments,the second antigen binding portion is fused to the first antigen bindingportion chemically. In some embodiments, the second antigen bindingportion is fused to the first antigen binding portion via a peptide bondor a peptide linker. In some embodiments, the peptide linker is no morethan about 30 (such as no more than about any one of 25, 20, or 15)amino acids long. In some embodiments, the first antigen bindingfragment comprises an Fc region, such as an IgG1 Fc.

In some embodiments, there is provided a method of treating aninflammatory or autoimmune disease in an individual in need thereof,comprising administering an effective amount of a pharmaceuticalcomposition comprising a multispecific (such as bispecific) antigenbinding protein and a pharmaceutically acceptable carrier, wherein theMABP comprises: (a) a first antigen binding portion comprising afull-length antibody (such as adalimumab) consisting of two heavy chainsand two light chains, wherein the full-length antibody specificallybinds TNF-α; and (b) a second antigen binding portion comprising an sdAbthat specifically binds IL-1β, wherein the first antigen binding portionand the second antigen binding portion are fused to each other. In someembodiments, the sdAb is a camelid, humanized, or human sdAb. In someembodiments, the inflammatory or autoimmune disease is selected from thegroup consisting of arthritis (such as rheumatoid arthritis, juvenileidiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, andarthritic ulcerative colitis), colitis, psoriasis, severe asthma, andmoderate to severe Cronh's disease. In some embodiments, the secondantigen binding portion is fused to the first antigen binding portion atthe N-terminus of one or each of the two heavy chains, the N-terminus ofone or each of the two light chains, the N-terminus of the Fc region,the C-terminus of one or each of the two heavy chains, or the C-terminusof one or each of the two light chains. In some embodiments, the secondantigen binding portion is fused to the first antigen binding portionchemically. In some embodiments, the second antigen binding portion isfused to the first antigen binding portion via a peptide bond or apeptide linker. In some embodiments, the peptide linker is no more thanabout 30 (such as no more than about any one of 25, 20, or 15) aminoacids long. In some embodiments, the first antigen binding fragmentcomprises an Fc region, such as an IgG1 Fc.

In some embodiments, there is provided a method of treating aninflammatory or autoimmune disease in an individual in need thereof,comprising administering an effective amount of a pharmaceuticalcomposition comprising a multispecific (such as bispecific) antigenbinding protein and a pharmaceutically acceptable carrier, wherein theMABP comprises: (a) a first antigen binding portion comprising afull-length antibody (such as mepolizumab) consisting of two heavychains and two light chains, wherein the full-length antibodyspecifically binds IL-5; and (b) a second antigen binding portioncomprising an sdAb that specifically binds eotaxin-1, wherein the firstantigen binding portion and the second antigen binding portion are fusedto each other. In some embodiments, the sdAb is a camelid, humanized, orhuman sdAb. In some embodiments, the inflammatory or autoimmune diseaseis selected from the group consisting of arthritis (such as rheumatoidarthritis, juvenile idiopathic arthritis, psoriatic arthritis,ankylosing spondylitis, and arthritic ulcerative colitis), colitis,psoriasis, severe asthma, and moderate to severe Cronh's disease. Insome embodiments, the second antigen binding portion is fused to thefirst antigen binding portion at the N-terminus of one or each of thetwo heavy chains, the N-terminus of one or each of the two light chains,the N-terminus of the Fc region, the C-terminus of one or each of thetwo heavy chains, or the C-terminus of one or each of the two lightchains. In some embodiments, the second antigen binding portion is fusedto the first antigen binding portion chemically. In some embodiments,the second antigen binding portion is fused to the first antigen bindingportion via a peptide bond or a peptide linker. In some embodiments, thepeptide linker is no more than about 30 (such as no more than about anyone of 25, 20, or 15) amino acids long. In some embodiments, the firstantigen binding fragment comprises an Fc region, such as an IgG1 Fc.

Dosage and Routes of Administration

Dosages and desired drug concentrations of pharmaceutical compositionsof the present application may vary depending on the particular useenvisioned. The determination of the appropriate dosage or route ofadministration is well within the skill of an ordinary artisan. Animalexperiments provide reliable guidance for the determination of effectivedoses for human therapy. Interspecies scaling of effective doses can beperformed following the principles laid down by Mordenti, J. andChappell, W. “The Use of Interspecies Scaling in Toxicokinetics,” InToxicokinetics and New Drug Development, Yacobi et al., Eds, PergamonPress, New York 1989, pp. 42-46.

When in vivo administration of the MABPs described herein are used,normal dosage amounts may vary from about 10 ng/kg up to about 100 mg/kgof mammal body weight or more per day, preferably about 1 mg/kg/day to10 mg/kg/day, depending upon the route of administration. It is withinthe scope of the present application that different formulations will beeffective for different treatments and different disorders, and thatadministration intended to treat a specific organ or tissue maynecessitate delivery in a manner different from that to another organ ortissue. Moreover, dosages may be administered by one or more separateadministrations, or by continuous infusion. For repeated administrationsover several days or longer, depending on the condition, the treatmentis sustained until a desired suppression of disease symptoms occurs.However, other dosage regimens may be useful. The progress of thistherapy is easily monitored by conventional techniques and assays.

In some embodiments, the pharmaceutical composition is administered fora single time. In some embodiments, the pharmaceutical composition isadministered for multiple times (such as any of 2, 3, 4, 5, 6, or moretimes). In some embodiments, the pharmaceutical composition isadministered once per week, once 2 weeks, once 3 weeks, once 4 weeks,once per month, once per 2 months, once per 3 months, once per 4 months,once per 5 months, once per 6 months, once per 7 months, once per 8months, once per 9 months, or once per year. In some embodiments, theinterval between administrations is about any one of 1 week to 2 weeks,2 weeks to 1 month, 2 weeks to 2 months, 1 month to 2 months, 1 month to3 months, 3 months to 6 months, or 6 months to a year. The optimaldosage and treatment regime for a particular patient can readily bedetermined by one skilled in the art of medicine by monitoring thepatient for signs of disease and adjusting the treatment accordingly.

The pharmaceutical compositions of the present application, includingbut not limited to reconstituted and liquid formulations, areadministered to an individual in need of treatment with the MABPs,preferably a human, in accord with known methods, such as intravenousadministration as a bolus or by continuous infusion over a period oftime, by intramuscular, intraperitoneal, intracerobrospinal,subcutaneous, intra-articular, intrasynovial, intrathecal, oral,topical, or inhalation routes.

In some embodiments, the pharmaceutical compositions are administered tothe individual by subcutaneous (i.e. beneath the skin) administration.For such purposes, the pharmaceutical compositions may be injected usinga syringe. However, other devices for administration of thepharmaceutical compositions are available such as injection devices;injector pens; auto-injector devices, needleless devices; andsubcutaneous patch delivery systems.

In some embodiments, the pharmaceutical compositions are administered tothe individual intravenously. In some embodiments, the pharmaceuticalcomposition is administered to an individual by infusion, such asintravenous infusion. Infusion techniques for immunotherapy are known inthe art (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676(1988)).

V. Methods of Preparation

The present application also provides isolated nucleic acids encodingthe MABPs, vectors and host cells comprising such isolated nucleicacids, and recombinant methods for the production of the MABPs.

For recombinant production of the MABP, the nucleic acids encoding thefull-length antibody or antigen binding fragment of the first antigenbinding portion, and the sdAb are isolated and inserted into areplicable vector for further cloning (amplification of the DNA) or forexpression. In some embodiments, the nucleic acid encoding thefull-length antibody or antigen binding fragment of the first antigenbinding portion is recombinantly fused to the nucleic acid encoding thesdAb of the second antigen binding portion and optionally via a nucleicacid encoding a peptide linker, all in frame for translation withrespect to each other to provide a nucleic acid encoding the MABP. DNAencoding the MABP, components thereof, or the sdAb is readily isolatedand sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of the antibody). Many vectors areavailable. The choice of vector depends in part on the host cell to beused. Generally, preferred host cells are of either prokaryotic oreukaryotic (generally mammalian) origin. Alternatively, the firstantigen binding fragment and the second antigen binding fragment areeach prepared recombinantly using prokaryotic or eukaryotic host cellscomprising nucleic acids that encode the first antigen binding fragmentand the second antigen binding fragment respectively. The expressedfirst antigen binding fragment and the second antigen binding fragmentare then conjugated chemically, and purified in order to provide theMABP.

1. Protein Production in Prokaryotic Cells a) Vector Construction

Polynucleotide sequences encoding polypeptide components of the MABP ofthe present application can be obtained using standard recombinanttechniques. Desired polynucleotide sequences may be isolated andsequenced from antibody producing cells such as hybridoma cells.Alternatively, polynucleotides can be synthesized using nucleotidesynthesizer or PCR techniques. Once obtained, sequences encoding thepolypeptides are inserted into a recombinant vector capable ofreplicating and expressing heterologous polynucleotides in prokaryotichosts. Many vectors that are available and known in the art can be usedfor the purpose of the present application. Selection of an appropriatevector will depend mainly on the size of the nucleic acids to beinserted into the vector and the particular host cell to be transformedwith the vector. Each vector contains various components, depending onits function (amplification or expression of heterologouspolynucleotide, or both) and its compatibility with the particular hostcell in which it resides. The vector components generally include, butare not limited to: an origin of replication, a selection marker gene, apromoter, a ribosome binding site (RBS), a signal sequence, theheterologous nucleic acid insert and a transcription terminationsequence.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with the host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as marking sequences which are capable of providingphenotypic selection in transformed cells. For example, E. coli istypically transformed using pBR322, a plasmid derived from an E. colispecies. pBR322 contains genes encoding ampicillin (Amp) andtetracycline (Tet) resistance and thus provides easy means foridentifying transformed cells. pBR322, its derivatives, or othermicrobial plasmids or bacteriophage may also contain, or be modified tocontain, promoters which can be used by the microbial organism forexpression of endogenous proteins. Examples of pBR322 derivatives usedfor expression of particular antibodies are described in detail inCarter et al., U.S. Pat. No. 5,648,237.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used astransforming vectors in connection with these hosts. For example,bacteriophage such as GEM™-11 may be utilized in making a recombinantvector which can be used to transform susceptible host cells such as E.coli LE392.

The expression vector described herein may comprise two or morepromoter-cistron pairs, encoding each of the polypeptide components. Apromoter is an untranslated regulatory sequence located upstream (5′) toa cistron that modulates its expression. Prokaryotic promoters typicallyfall into two classes, inducible and constitutive. Inducible promoter isa promoter that initiates increased levels of transcription of thecistron under its control in response to changes in the culturecondition, e.g. the presence or absence of a nutrient or a change intemperature.

A large number of promoters recognized by a variety of potential hostcells are well known. The selected promoter can be operably linked tocistron DNA encoding the light or heavy chain by removing the promoterfrom the source DNA via restriction enzyme digestion and inserting theisolated promoter sequence into the vector. Both the native promotersequence and many heterologous promoters may be used to directamplification and/or expression of the target genes. In someembodiments, heterologous promoters are utilized, as they generallypermit greater transcription and higher yields of expressed target geneas compared to the native target polypeptide promoter.

Promoters suitable for use with prokaryotic hosts include the PhoApromoter, the—galactamase and lactose promoter systems, a tryptophan(trp) promoter system and hybrid promoters such as the tac or the trcpromoter. However, other promoters that are functional in bacteria (suchas other known bacterial or phage promoters) are suitable as well. Theirnucleotide sequences have been published, thereby enabling a skilledworker operably to ligate them to cistrons encoding the target light andheavy chains (Siebenlist et al. (1980) Cell 20: 269) using linkers oradaptors to supply any required restriction sites.

In one aspect, each cistron within the recombinant vector comprises asecretion signal sequence component that directs translocation of theexpressed polypeptides across a membrane. In general, the signalsequence may be a component of the vector, or it may be a part of thetarget polypeptide DNA that is inserted into the vector. The signalsequence selected for the purpose of this application should be one thatis recognized and processed (i.e. cleaved by a signal peptidase) by thehost cell. For prokaryotic host cells that do not recognize and processthe signal sequences native to the heterologous polypeptides, the signalsequence is substituted by a prokaryotic signal sequence selected, forexample, from the group consisting of the alkaline phosphatase,penicillinase, Ipp, or heat-stable enterotoxin II (STII) leaders, LamB,PhoE, PelB, OmpA and MBP. In some embodiments, the signal sequences usedin both cistrons of the expression system are STII signal sequences orvariants thereof.

In some embodiments, the production of the MABPs can occur in thecytoplasm of the host cell, and therefore does not require the presenceof secretion signal sequences within each cistron. In some embodiments,polypeptide components, such as the polypeptide encoding the V_(H)domain of the first antigen binding portion optionally fused to thesecond antigen binding portion, and the polypeptide encoding the V_(L)domain of the first antigen binding portion optionally fused to thesecond antigen binding portion, are expressed, folded and assembled toform functional MABPs within the cytoplasm. Certain host strains (e.g.,the E. coli trxB⁻ strains) provide cytoplasm conditions that arefavorable for disulfide bond formation, thereby permitting properfolding and assembly of expressed protein subunits. Proba and PluckthunGene, 159:203 (1995).

The present application provides an expression system in which thequantitative ratio of expressed polypeptide components can be modulatedin order to maximize the yield of secreted and properly assembled theMABPs of the present application. Such modulation is accomplished atleast in part by simultaneously modulating translational strengths forthe polypeptide components. One technique for modulating translationalstrength is disclosed in Simmons et al., U.S. Pat. No. 5,840,523. Itutilizes variants of the translational initiation region (TIR) within acistron. For a given TIR, a series of amino acid or nucleic acidsequence variants can be created with a range of translationalstrengths, thereby providing a convenient means by which to adjust thisfactor for the desired expression level of the specific chain. TIRvariants can be generated by conventional mutagenesis techniques thatresult in codon changes which can alter the amino acid sequence,although silent changes in the nucleotide sequence are preferred.Alterations in the TIR can include, for example, alterations in thenumber or spacing of Shine-Dalgarno sequences, along with alterations inthe signal sequence. One method for generating mutant signal sequencesis the generation of a “codon bank” at the beginning of a codingsequence that does not change the amino acid sequence of the signalsequence (i.e., the changes are silent). This can be accomplished bychanging the third nucleotide position of each codon; additionally, someamino acids, such as leucine, serine, and arginine, have multiple firstand second positions that can add complexity in making the bank. Thismethod of mutagenesis is described in detail in Yansura et al. (1992)METHODS: A Companion to Methods in Enzymol. 4:151-158.

Preferably, a set of vectors is generated with a range of TIR strengthsfor each cistron therein. This limited set provides a comparison ofexpression levels of each chain as well as the yield of the desired MABPproducts under various TIR strength combinations. TIR strengths can bedetermined by quantifying the expression level of a reporter gene asdescribed in detail in Simmons et al. U.S. Pat. No. 5,840,523. Based onthe translational strength comparison, the desired individual TIRs areselected to be combined in the expression vector constructs of thepresent application.

b) Prokaryotic Host Cells.

Prokaryotic host cells suitable for expressing the MABPs of the presentapplication include Archaebacteria and Eubacteria, such as Gram-negativeor Gram-positive organisms. Examples of useful bacteria includeEscherichia (e.g., E. coli), Bacilli (e.g., B. subtilis),Enterobacteria, Pseudomonas species (e.g., P. aeruginosa), Salmonellatyphimurium, Serratia marcescans, Klebsiella, Proteus, Shigella,Rhizobia, Vitreoscilla, or Paracoccus. In one embodiment, gram-negativecells are used. In one embodiment, E. coli cells are used as hosts.Examples of E. coli strains include strain W3110 (Bachmann, Cellular andMolecular Biology, vol. 2 (Washington, D.C.: American Society forMicrobiology, 1987), pp. 1190-1219; ATCC Deposit No. 27,325) andderivatives thereof, including strain 33D3 having genotype W3110 AfhuA(AtonA) ptr3 lac Iq lacL8 AompT A (nmpc-fepE) degP41 kan^(R) (U.S. Pat.No. 5,639,635). Other strains and derivatives thereof, such as E. coli294 (ATCC 31,446), E. coli B, E. coli 1776 (ATCC 31,537) and E. coliRV308 (ATCC 31,608) are also suitable. These examples are illustrativerather than limiting. Methods for constructing derivatives of any of theabove-mentioned bacteria having defined genotypes are known in the artand described in, for example, Bass et al., Proteins, 8:309-314 (1990).It is generally necessary to select the appropriate bacteria taking intoconsideration replicability of the replicon in the cells of a bacterium.For example, E. coli, Serratia, or Salmonella species can be suitablyused as the host when well known plasmids such as pBR322, pBR325,pACYC177, or pKN410 are used to supply the replicon.

Typically the host cell should secrete minimal amounts of proteolyticenzymes, and additional protease inhibitors may desirably beincorporated in the cell culture.

c) Protein Production

Host cells are transformed with the above-described expression vectorsand cultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences. Transformation means introducing DNAinto the prokaryotic host so that the DNA is replicable, either as anextrachromosomal element or by chromosomal integrant. Depending on thehost cell used, transformation is done using standard techniquesappropriate to such cells. The calcium treatment employing calciumchloride is generally used for bacterial cells that contain substantialcell-wall barriers. Another method for transformation employspolyethylene glycol/DMSO. Yet another technique used is electroporation.

Prokaryotic cells used to produce the MABPs of the present applicationare grown in media known in the art and suitable for culture of theselected host cells. Examples of suitable media include luria broth (LB)plus necessary nutrient supplements. In some embodiments, the media alsocontains a selection agent, chosen based on the construction of theexpression vector, to selectively permit growth of prokaryotic cellscontaining the expression vector. For example, ampicillin is added tomedia for growth of cells expressing ampicillin resistant gene.

Any necessary supplements besides carbon, nitrogen, and inorganicphosphate sources may also be included at appropriate concentrationsintroduced alone or as a mixture with another supplement or medium suchas a complex nitrogen source. Optionally the culture medium may containone or more reducing agents selected from the group consisting ofglutathione, cysteine, cystamine, thioglycollate, dithioerythritol anddithiothreitol.

The prokaryotic host cells are cultured at suitable temperatures. For E.coli growth, for example, the preferred temperature ranges from about20° C. to about 39° C., more preferably from about 25° C. to about 37°C., even more preferably at about 30° C. The pH of the medium may be anypH ranging from about 5 to about 9, depending mainly on the hostorganism. For E. coli, the pH is preferably from about 6.8 to about 7.4,and more preferably about 7.0.

If an inducible promoter is used in the expression vector, proteinexpression is induced under conditions suitable for the activation ofthe promoter. In some embodiments, PhoA promoters are used forcontrolling transcription of the polypeptides. Accordingly, thetransformed host cells are cultured in a phosphate-limiting medium forinduction. Preferably, the phosphate-limiting medium is the C.R.A.Pmedium (see, e.g., Simmons et al., J. Immunol. Methods (2002),263:133-147). A variety of other inducers may be used, according to thevector construct employed, as is known in the art.

The expressed MABPs of the present application are secreted into andrecovered from the periplasm of the host cells. Protein recoverytypically involves disrupting the microorganism, generally by such meansas osmotic shock, sonication or lysis. Once cells are disrupted, celldebris or whole cells may be removed by centrifugation or filtration.The proteins may be further purified, for example, by affinity resinchromatography. Alternatively, proteins can be transported into theculture media and isolated therein. Cells may be removed from theculture and the culture supernatant being filtered and concentrated forfurther purification of the proteins produced. The expressedpolypeptides can be further isolated and identified using commonly knownmethods such as polyacrylamide gel electrophoresis (PAGE) and Westernblot assay.

Alternatively, protein production is conducted in large quantity by afermentation process. Various large-scale fed-batch fermentationprocedures are available for production of recombinant proteins.Large-scale fermentations have at least 1000 liters of capacity,preferably about 1,000 to 100,000 liters of capacity. These fermentorsuse agitator impellers to distribute oxygen and nutrients, especiallyglucose (the preferred carbon/energy source). Small scale fermentationrefers generally to fermentation in a fermentor that is no more thanapproximately 100 liters in volumetric capacity, and can range fromabout 1 liter to about 100 liters.

During the fermentation process, induction of protein expression istypically initiated after the cells have been grown under suitableconditions to a desired density, e.g., an OD₅₅₀ of about 180-220, atwhich stage the cells are in the early stationary phase. A variety ofinducers may be used, according to the vector construct employed, as isknown in the art and described above. Cells may be grown for shorterperiods prior to induction. Cells are usually induced for about 12-50hours, although longer or shorter induction time may be used.

To improve the production yield and quality of the MABPs of the presentapplication, various fermentation conditions can be modified. Forexample, to improve the proper assembly and folding of the secretedpolypeptides, additional vectors overexpressing chaperone proteins, suchas Dsb proteins (DsbA, DsbB, DsbC, DsbD and or DsbG) or FkpA (apeptidylprolyl cis,trans-isomerase with chaperone activity) can be usedto co-transform the host prokaryotic cells. The chaperone proteins havebeen demonstrated to facilitate the proper folding and solubility ofheterologous proteins produced in bacterial host cells. Chen et al.(1999) J Bio Chem 274:19601-19605; Georgiou et al., U.S. Pat. No.6,083,715; Georgiou et al., U.S. Pat. No. 6,027,888; Bothmann andPluckthun (2000) J. Biol. Chem. 275:17100-17105; Ramm and Pluckthun(2000) J. Biol. Chem. 275:17106-17113; Arie et al. (2001) Mol.Microbiol. 39:199-210.

To minimize proteolysis of expressed heterologous proteins (especiallythose that are proteolytically sensitive), certain host strainsdeficient for proteolytic enzymes can be used for the presentapplication. For example, host cell strains may be modified to effectgenetic mutation(s) in the genes encoding known bacterial proteases suchas Protease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V,Protease VI and combinations thereof. Some E. coli protease-deficientstrains are available and described in, for example, Joly et al. (1998),supra; Georgiou et al., U.S. Pat. No. 5,264,365; Georgiou et al., U.S.Pat. No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72(1996).

E. coli strains deficient for proteolytic enzymes and transformed withplasmids overexpressing one or more chaperone proteins may be used ashost cells in the expression system encoding the MABPs of the presentapplication.

d) Protein Purification

The MABPs produced herein are further purified to obtain preparationsthat are substantially homogeneous for further assays and uses. Standardprotein purification methods known in the art can be employed. Thefollowing procedures are exemplary of suitable purification procedures:fractionation on immunoaffinity or ion-exchange columns, ethanolprecipitation, reverse phase HPLC, chromatography on silica or on acation-exchange resin such as DEAE, chromatofocusing, SDS-PAGE, ammoniumsulfate precipitation, and gel filtration using, for example, SephadexG-75.

In some embodiments, Protein A immobilized on a solid phase is used forimmunoaffinity purification of the MABPs comprising an Fc regiondescribed herein. Protein A is a 411 (D cell wall protein fromStaphylococcus aureas which binds with a high affinity to the Fc regionof antibodies. Lindmark et al (1983) J. Immunol. Meth. 62:1-13. Thesolid phase to which Protein A is immobilized is preferably a columncomprising a glass or silica surface, more preferably a controlled poreglass column or a silicic acid column. In some applications, the columnhas been coated with a reagent, such as glycerol, in an attempt toprevent nonspecific adherence of contaminants. The solid phase is thenwashed to remove contaminants non-specifically bound to the solid phase.Finally the MABPs of interest are recovered from the solid phase byelution.

2. Protein Production in Eukaryotic Cells

For Eukaryotic expression, the vector components generally include, butare not limited to, one or more of the following, a signal sequence, anorigin of replication, one or more marker genes, and enhancer element, apromoter, and a transcription termination sequence.

a) Signal Sequence Component

A vector for use in a eukaryotic host may also an insert that encodes asignal sequence or other polypeptide having a specific cleavage site atthe N-terminus of the mature protein or polypeptide. The heterologoussignal sequence selected preferably is one that is recognized andprocessed (i.e., cleaved by a signal peptidase) by the host cell. Inmammalian cell expression, mammalian signal sequences as well as viralsecretory leaders, for example, the herpes simplex gD signal, areavailable.

The DNA for such precursor region is ligated in reading frame to DNAencoding the MABPs of the present application.

b) Origin of Replication

Generally, the origin of replication component is not needed formammalian expression vectors (the SV40 origin may typically be used onlybecause it contains the early promoter).

c) Selection Gene Component

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take upnucleic acid encoding the MABPs of the present application, such asDHFR, thymidine kinase, metallothionein-I and -II, preferably primatemetallothionein genes, adenosine deaminase, ornithine decarboxylase,etc.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity (e.g., ATCCCRL-9096).

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with the polypeptideencoding-DNA sequences, wild-type DHFR protein, and another selectablemarker such as aminoglycoside 3′-phosphotransferase (APH) can beselected by cell growth in medium containing a selection agent for theselectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

d) Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the nucleicacid encoding the desired polypeptide sequences. Virtually alleukaryotic genes have an AT-rich region located approximately 25 to 30based upstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of thetranscription of many genes is a CNCAAT region where N may be anynucleotide. A the 3′ end of most eukaryotic is an AATAAA sequence thatmay be the signal for addition of the poly A tail to the 3′ end of thecoding sequence. All of these sequences may be inserted into eukaryoticexpression vectors.

Other promoters suitable for use with prokaryotic hosts include the phoApromoter, -lactamase and lactose promoter systems, alkaline phosphatasepromoter, a tryptophan (trp) promoter system, and hybrid promoters suchas the tac promoter. However, other known bacterial promoters aresuitable. Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encoding theMABPs.

Polypeptide transcription from vectors in mammalian host cells iscontrolled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus and most preferablySimian Virus 40 (SV40), from heterologous mammalian promoters, e.g., theactin promoter or an immunoglobulin promoter, from heat-shock promoters,provided such promoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., Nature 297:598-601 (1982) onexpression of human-interferon cDNA in mouse cells under the control ofa thymidine kinase promoter from herpes simplex virus. Alternatively,the Rous Sarcoma Virus long terminal repeat can be used as the promoter.

e) Enhancer Element Component

Transcription of a DNA encoding the MABPs of the present application byhigher eukaryotes is often increased by inserting an enhancer sequenceinto the vector. Many enhancer sequences are now known from mammaliangenes (globin, elastase, albumin, α-fetoprotein, and insulin).Typically, however, one will use an enhancer from a eukaryotic cellvirus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18(1982) on enhancing elements for activation of eukaryotic promoters. Theenhancer may be spliced into the vector at a position 5′ or 3′ to thepolypeptide encoding sequence, but is preferably located at a site 5′from the promoter.

f) Transcription Termination Component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the polypeptide-encoding mRNA. One useful transcriptiontermination component is the bovine growth hormone polyadenylationregion. See WO94/11026 and the expression vector disclosed therein.

a) Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein include higher eukaryote cells described herein, includingvertebrate host cells. Propagation of vertebrate cells in culture(tissue culture) has become a routine procedure. Examples of usefulmammalian host cell lines are monkey kidney CV1 line transformed by SV40(COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cellssubcloned for growth in suspension culture, Graham et al., J. Gen Virol.36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinesehamster ovary cells/−DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci.USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinomacells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals N.Y. Acad.Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatomaline (Hep G2).

Host cells are transformed with the above-described expression orcloning vectors for MABPs production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

h) Culturing the Host Cells

The host cells used to produce the MABPs of the present application maybe cultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium (MEM), (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium (DMEM), Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal.Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762;4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re.30,985 may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleotides (such as adenosine and thymidine),antibiotics (such as GENTAMYCIN™ drug), trace elements (defined asinorganic compounds usually present at final concentrations in themicromolar range), and glucose or an equivalent energy source. Any othernecessary supplements may also be included at appropriate concentrationsthat would be known to those skilled in the art. The culture conditions,such as temperature, pH, and the like, are those previously used withthe host cell selected for expression, and will be apparent to theordinarily skilled artisan.

i) Protein Purification

When using recombinant techniques, the MABPs can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the MABP or the sdAb is produced intracellularly, as a firststep, the particulate debris, either host cells or lysed fragments, areremoved, for example, by centrifugation or ultrafiltration. Carter etal., Bio/Technology 10:163-167 (1992) describe a procedure for isolatingantibodies which are secreted to the periplasmic space of E. coli.Briefly, cell paste is thawed in the presence of sodium acetate (pH3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.Cell debris can be removed by centrifugation. Where the MABP or the sdAbis secreted into the medium, supernatants from such expression systemsare generally first concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. A protease inhibitor such as PMSF may be includedin any of the foregoing steps to inhibit proteolysis and antibiotics maybe included to prevent the growth of adventitious contaminants.

The protein composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the MABP. Protein A can beused to purify the MABPs that are based on human immunoglobulinscontaining 1, 2, or 4 heavy chains (Lindmark et al., J. Immunol. Meth.62:1-13 (1983)). Protein G is recommended for all mouse isotypes and forhuman 3 (Guss et al., EMBO J. 5:15671575 (1986)). The matrix to whichthe affinity ligand is attached is most often agarose, but othermatrices are available. Mechanically stable matrices such as controlledpore glass or poly(styrene-divinyl)benzene allow for faster flow ratesand shorter processing times than can be achieved with agarose. Wherethe MABP comprises a C_(H)3 domain, the Bakerbond ABX™ resin (J. T.Baker, Phillipsburg, N.J.) is useful for purification. Other techniquesfor protein purification such as fractionation on an ion-exchangecolumn, ethanol precipitation, Reverse Phase HPLC, chromatography onsilica, chromatography on heparin SEPHAROSE™ chromatography on an anionor cation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the MABP or the sdAb to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe MABP or the sdAb of interest and contaminants may be subjected tolow pH hydrophobic interaction chromatography using an elution buffer ata pH between about 2.5-4.5, preferably performed at low saltconcentrations (e.g., from about 0-0.25M salt).

3. Antibody Production

Components of the MABPs, such as conventional 4-chain antibodies,antigen-binding fragments, and sdAbs, can be produced using any knownmethods in the art, including methods described below.

The sdAbs (such as V_(H)Hs) may be obtained using methods known in theart such as by immunizing a Camelidae species (such as camel or llama)and obtaining hybridomas therefrom, or by cloning a library of sdAbsusing molecular biology techniques known in the art and subsequentselection by ELISA with individual clones of unselected libraries or byusing phage display.

1) Monoclonal Antibodies

Monoclonal antibodies are obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally occurringmutations and/or post-translational modifications (e.g., isomerizations,amidations) that may be present in minor amounts. Thus, the modifier“monoclonal” indicates the character of the antibody as not being amixture of discrete antibodies.

For example, the monoclonal antibodies may be made using the hybridomamethod first described by Kohler et al., Nature, 256:495 (1975), or maybe made by recombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized as hereinabove described to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind the protein used for immunization. Alternatively,lymphocytes may be immunized in vitro. Lymphocytes then are fused withmyeloma cells using a suitable fusing agent, such as polyethyleneglycol, to form a hybridoma cell (Goding, Monoclonal Antibodies:Principles and Practice, pp. 59-103 (Academic Press, 1986).

The immunizing agent will typically include the antigenic protein or afusion variant thereof. Generally either peripheral blood lymphocytes(“PBLs”) are used if cells of human origin are desired, or spleen cellsor lymph node cells are used if non-human mammalian sources are desired.The lymphocytes are then fused with an immortalized cell line using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell. Goding, Monoclonal Antibodies: Principles and Practice, AcademicPress (1986), pp. 59-103.

Immortalized cell lines are usually transformed mammalian cells,particularly myeloma cells of rodent, bovine and human origin. Usually,rat or mouse myeloma cell lines are employed. The hybridoma cells thusprepared are seeded and grown in a suitable culture medium thatpreferably contains one or more substances that inhibit the growth orsurvival of the unfused, parental myeloma cells. For example, if theparental myeloma cells lack the enzyme hypoxanthine guaninephosphoribosyl transferase (HGPRT or HPRT), the culture medium for thehybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which are substances that prevent the growth ofHGPRT-deficient cells.

Preferred immortalized myeloma cells are those that fuse efficiently,support stable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. Among these, preferred are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2cells (and derivatives thereof, e.g., X63-Ag8-653) available from theAmerican Type Culture Collection, Manassas, Va. USA. Human myeloma andmouse-human heteromyeloma cell lines also have been described for theproduction of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001(1984); Brodeur et al., Monoclonal Antibody Production Techniques andApplications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunosorbent assay (ELISA).

The culture medium in which the hybridoma cells are cultured can beassayed for the presence of monoclonal antibodies directed against thedesired antigen. Preferably, the binding affinity and specificity of themonoclonal antibody can be determined by immunoprecipitation or by an invitro binding assay, such as radioimmunoassay (RIA) or enzyme-linkedassay (ELISA). Such techniques and assays are known in the in art. Forexample, binding affinity may be determined by the Scatchard analysis ofMunson et al., Anal. Biochem., 107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, supra). Suitable culture media for this purpose include, forexample, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells maybe grown in vivo as tumors in a mammal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

Monoclonal antibodies may also be made by recombinant DNA methods, suchas those described in U.S. Pat. No. 4,816,567, and as described above.DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of murine antibodies). The hybridoma cells serve as apreferred source of such DNA. Once isolated, the DNA may be placed intoexpression vectors, which are then transfected into host cells such asE. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, ormyeloma cells that do not otherwise produce immunoglobulin protein, inorder to synthesize monoclonal antibodies in such recombinant hostcells. Review articles on recombinant expression in bacteria of DNAencoding the antibody include Skerra et al., Curr. Opinion in Immunol.,5:256-262 (1993) and Plückthun, Immunol. Revs. 130:151-188 (1992).

In a further embodiment, antibodies can be isolated from antibody phagelibraries generated using the techniques described in McCafferty et al.,Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991)and Marks et al., J. Mol. Biol., 222:581-597 (1991) describe theisolation of murine and human antibodies, respectively, using phagelibraries. Subsequent publications describe the production of highaffinity (nM range) human antibodies by chain shuffling (Marks et al.,Bio/Technology, 10:779-783 (1992)), as well as combinatorial infectionand in vivo recombination as a strategy for constructing very largephage libraries (Waterhouse et al., Nucl. Acids Res., 21:2265-2266(1993)). Thus, these techniques are viable alternatives to traditionalmonoclonal antibody hybridoma techniques for isolation of monoclonalantibodies.

The DNA also may be modified, for example, by substituting the codingsequence for human heavy- and light-chain constant domains in place ofthe homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison, etal., Proc. Natl Acad. Sci. USA, 81:6851 (1984)), or by covalentlyjoining to the immunoglobulin coding sequence all or part of the codingsequence for a non-immunoglobulin polypeptide. Typically suchnon-immunoglobulin polypeptides are substituted for the constant domainsof an antibody, or they are substituted for the variable domains of oneantigen-combining site of an antibody to create a chimeric bivalentantibody comprising one antigen-combining site having specificity for anantigen and another antigen-combining site having specificity for adifferent antigen.

The monoclonal antibodies described herein may by monovalent, thepreparation of which is well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain and amodified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues may be substituted withanother amino acid residue or are deleted so as to prevent crosslinking.In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly Fabfragments, can be accomplished using routine techniques known in theart.

Chimeric or hybrid antibodies also may be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins may be constructed usinga disulfide-exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

2) Humanized Antibodies

The antibodies may further comprise humanized or human antibodies.Humanized forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.Humanized antibodies include human immunoglobulins (recipient antibody)in which residues from a complementarity determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity and capacity. In some instances, Fv frameworkresidues of the human immunoglobulin are replaced by correspondingnon-human residues. Humanized antibodies may also comprise residueswhich are found neither in the recipient antibody nor in the importedCDR or framework sequences. In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomain, in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin consensus sequence.The humanized antibody optimally also will comprise at least a portionof an immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. Jones et al., Nature 321: 522-525 (1986); Riechmann etal., Nature 332: 323-329 (1988) and Presta, Curr. Opin. Struct. Biol. 2:593-596 (1992).

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers,Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988), orthrough substituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework (FR) for the humanized antibody. Sims et al., J.Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901(1987). Another method uses a particular framework derived from theconsensus sequence of all human antibodies of a particular subgroup oflight or heavy chains. The same framework may be used for severaldifferent humanized antibodies. Carter et al., Proc. Natl. Acad. Sci.USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.

Various forms of the humanized antibody are contemplated. For example,the humanized antibody may be an antibody fragment, such as an Fab,which is optionally conjugated with one or more cytotoxic agent(s) inorder to generate an immunoconjugate. Alternatively, the humanizedantibody may be an intact antibody, such as an intact IgG1 antibody.

In some embodiments, the sdAbs are modified, such as humanized, withoutdiminishing the native affinity of the domain for antigen and whilereducing its immunogenicity with respect to a heterologous species. Forexample, the amino acid residues of the antibody variable domain(V_(H)H) of an llama antibody can be determined, and one or more of theCamelidae amino acids, for example, in the framework regions, arereplaced by their human counterpart as found in the human consensussequence, without that polypeptide losing its typical character, i.e.the humanization does not significantly affect the antigen bindingcapacity of the resulting polypeptide. Humanization of Camelidae sdAbsrequires the introduction and mutagenesis of a limited amount of aminoacids in a single polypeptide chain. This is in contrast to humanizationof scFv, Fab′, (Fab′)₂ and IgG, which requires the introduction of aminoacid changes in two chains, the light and the heavy chain and thepreservation of the assembly of both chains.

3) Human Antibodies

As an alternative to humanization, human antibodies can be generated.For example, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551(1993); Jakobovits et al., Nature. 362:255-258 (1993); Bruggermann etal., Year in Immuno., 7:33 (1993); U.S. Pat. No. 5,591,669 and WO97/17852. Transgenic mice or rats capable of producing fully human sdAbsare known in the art. See, e.g., US20090307787A1, U.S. Pat. No.8,754,287, US20150289489A1, US20100122358A1, and WO2004049794.

Alternatively, phage display technology can be used to produce humanantibodies and antibody fragments in vitro, from immunoglobulin variable(V) domain gene repertoires from unimmunized donors. McCafferty et al.,Nature 348:552-553 (1990); Hoogenboom and Winter, J. Mol. Biol. 227: 381(1991). According to this technique, antibody V domain genes are clonedin-frame into either a major or minor coat protein gene of a filamentousbacteriophage, such as M13 or fd, and displayed as functional antibodyfragments on the surface of the phage particle. Because the filamentousparticle contains a single-stranded DNA copy of the phage genome,selections based on the functional properties of the antibody alsoresult in selection of the gene encoding the antibody exhibiting thoseproperties. Thus, the phage mimics some of the properties of the B-cell.Phage display can be performed in a variety of formats, reviewed in,e.g., Johnson, Kevin S, and Chiswell, David J., Curr. Opin Struct. Biol.3:564-571 (1993). Several sources of V-gene segments can be used forphage display. Clackson et al., Nature 352:624-628 (1991) isolated adiverse array of anti-oxazolone antibodies from a small randomcombinatorial library of V genes derived from the spleens of immunizedmice. A repertoire of V genes from unimmunized human donors can beconstructed and antibodies to a diverse array of antigens (includingself-antigens) can be isolated essentially following the techniquesdescribed by Marks et al., J. Mol. Biol. 222:581-597 (1991), or Griffithet al., EMBO J. 12:725-734 (1993). See also, U.S. Pat. Nos. 5,565,332and 5,573,905.

The techniques of Cole et al., and Boerner et al., are also availablefor the preparation of human monoclonal antibodies (Cole et al.,Monoclonal Antibodies and Cancer Therapy, Alan R Liss, p. 77 (1985) andBoerner et al., J. Immunol. 147(1): 86-95 (1991). Similarly, humanantibodies can be made by introducing human immunoglobulin loci intotransgenic animals, e.g., mice in which the endogenous immunoglobulingenes have been partially or completely inactivated. Upon challenge,human antibody production is observed, which closely resembles that seenin humans in all respects, including gene rearrangement, assembly andantibody repertoire. This approach is described, for example, in U.S.Pat. Nos. 5,545,807; 5,545,806, 5,569,825, 5,625,126, 5,633,425,5,661,016 and in the following scientific publications: Marks et al.,Bio/Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859(1994); Morrison, Nature 368: 812-13 (1994), Fishwild et al., NatureBiotechnology 14: 845-51 (1996), Neuberger, Nature Biotechnology 14: 826(1996) and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).

Finally, human antibodies may also be generated in vitro by activated Bcells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).

4) Antibody Fragments

In certain circumstances there are advantages to using antibodyfragments, such as antigen binding fragments, rather than wholeantibodies. Smaller fragment sizes allow for rapid clearance, and maylead to improved access to solid tumors.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., J BiochemBiophys. Method. 24:107-117 (1992); and Brennan et al., Science 229:81(1985)). However, these fragments can now be produced directly byrecombinant host cells. Fab, Fv and scFv antibody fragments can all beexpressed in and secreted from E. coli, thus allowing the facileproduction of large amounts of these fragments. Antibody fragments canbe isolated from the antibody phage libraries discussed above.Alternatively, Fab′-SH fragments can be directly recovered from E. coliand chemically coupled to form F(ab′)₂ fragments (Carter et al.,Bio/Technology 10:163-167 (1992)). According to another approach,F(ab′)₂ fragments can be isolated directly from recombinant host cellculture. Fab and F(ab′)₂ with increase in vivo half-life is described inU.S. Pat. No. 5,869,046. In other embodiments, the antibody of choice isa single chain Fv fragment (scFv). See WO 93/16185; U.S. Pat. Nos.5,571,894 and 5,587,458. The antibody fragment may also be a “linearantibody”, e.g., as described in U.S. Pat. No. 5,641,870. Such linearantibody fragments may be monospecific or bispecific.

5) Bispecific and Multispecific Antibodies

Bispecific antibodies (BsAbs) are antibodies that have bindingspecificities for at least two different epitopes, including those onthe same or another protein. Alternatively, one arm can bind the targetantigen, and another arm can be combined with an arm that binds atriggering molecule on a leukocyte such as a T-cell receptor molecule(e.g., CD3), or Fc receptors for IgG (FcγR) such as FcγR1 (CD64), FcγRII(CD32) and FcγRIII (CD16), so as to focus and localize cellular defensemechanisms to the target antigen-expressing cell. Such antibodies can bederived from full length antibodies or antibody fragments (e.g. F(ab′)₂bispecific antibodies).

Bispecific antibodies may also be used to localize cytotoxic agents tocells which express the target antigen. Such antibodies possess one armthat binds the desired antigen and another arm that binds the cytotoxicagent (e.g., saporin, anti-interferon-α, vinca alkoloid, ricin A chain,methotrexate or radioactive isotope hapten). Examples of knownbispecific antibodies include anti-ErbB2/anti-FcgRIII (WO 96/16673),anti-ErbB2/anti-FcgRI (U.S. Pat. No. 5,837,234), anti-ErbB2/anti-CD3(U.S. Pat. No. 5,821,337).

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy-chain/light chain pairs,where the two chains have different specificities. Millstein et al.,Nature, 305:537-539 (1983). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829 and in Traunecker et al., EMBOJ., 10:3655-3659 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion preferablyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge, C_(H)2, and C_(H)3 regions. It is preferred tohave the first heavy-chain constant region (C_(H)1) containing the sitenecessary for light chain binding, present in at least one of thefusions. DNAs encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains in oneexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios are of noparticular significance.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecules provides for an easy way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies, see, for example, Suresh etal., Methods in Enzymology 121: 210 (1986).

According to another approach described in WO 96/27011 or U.S. Pat. No.5,731,168, the interface between a pair of antibody molecules can beengineered to maximize the percentage of heterodimers which arerecovered from recombinant cell culture. The preferred interfacecomprises at least a part of the C_(H)3 region of an antibody constantdomain. In this method, one or more small amino acid side chains fromthe interface of the first antibody molecule are replaced with largerside chains (e.g., tyrosine or tryptophan). Compensatory “cavities” ofidentical or similar size to the large side chains(s) are created on theinterface of the second antibody molecule by replacing large amino acidside chains with smaller ones (e.g., alanine or threonine). Thisprovides a mechanism for increasing the yield of the heterodimer overother unwanted end-products such as homodimers.

Techniques for generating bispecific antibodies from antibody fragmentshave been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)₂ fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-TNB derivative to form the bispecificantibody. The bispecific antibodies produced can be used as agents forthe selective immobilization of enzymes.

Fab′ fragments may be directly recovered from E. coli and chemicallycoupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:217-225 (1992) describes the production of fully humanized bispecificantibody F(ab′)₂ molecules. Each Fab′ fragment was separately secretedfrom E. coli and subjected to directed chemical coupling in vitro toform the bispecific antibody. The bispecific antibody thus formed wasable to bind cells overexpressing the ErbB2 receptor and normal human Tcells, as well as trigger the lytic activity of human cytotoxiclymphocytes against human breast tumor targets.

Various techniques for making and isolating bivalent antibody fragmentsdirectly from recombinant cell culture have also been described. Forexample, bivalent heterodimers have been produced using leucine zippers.Kostelny et al., J. Immunol., 148(5):1547-1553 (1992). The leucinezipper peptides from the Fos and Jun proteins were linked to the Fab′portions of two different antibodies by gene fusion. The antibodyhomodimers were reduced at the hinge region to form monomers and thenre-oxidized to form the antibody heterodimers. The “diabody” technologydescribed by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448(1993) has provided an alternative mechanism for makingbispecific/bivalent antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific/bivalentantibody fragments by the use of single-chain Fv (scFv) dimers has alsobeen reported. See Gruber et al., J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147: 60(1991).

Exemplary bispecific antibodies may bind two different epitopes on agiven molecule. Alternatively, an anti-protein arm may be combined withan arm which binds a triggering molecule on a leukocyte such as a T-cellreceptor molecule (e.g., CD2, CD3, CD28 or B7), or Fc receptors for IgG(FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) so as tofocus cellular defense mechanisms to the cell expressing the particularprotein. Bispecific antibodies may also be used to localize cytotoxicagents to cells which express a particular protein. Such antibodiespossess a protein-binding arm and an arm which binds a cytotoxic agentor a radionuclide chelator, such as EOTUBE, DPTA, DOTA or TETA. Anotherbispecific antibody of interest binds the protein of interest andfurther binds tissue factor (TF).

6) Multivalent Antibodies

A multivalent antibody may be internalized (and/or catabolized) fasterthan a bivalent antibody by a cell expressing an antigen to which theantibodies bind. The antibodies used as the first antigen bindingportion in the MABPs of the present application can be multivalentantibodies (which are other than of the IgM class) with three or moreantigen binding sites (e.g. tetravalent antibodies), which can bereadily produced by recombinant expression of nucleic acid encoding thepolypeptide chains of the antibody. The multivalent antibody cancomprise a dimerization domain and three or more antigen binding sites.The preferred dimerization domain comprises (or consists of) an Fcregion or a hinge region. In this scenario, the antibody will comprisean Fc region and three or more antigen binding sites amino-terminal tothe Fc region. The preferred multivalent antibody herein comprises (orconsists of) three to about eight, but preferably four, antigen bindingsites. The multivalent antibody comprises at least one polypeptide chain(and preferably two polypeptide chains), wherein the polypeptidechain(s) comprise two or more variable domains. For instance, thepolypeptide chain(s) may comprise VD1-(X1)_(n)-VD2-(X₂)_(n)-Fc, whereinVD1 is a first variable domain, VD2 is a second variable domain, Fc isone polypeptide chain of an Fc region, X1 and X2 represent an amino acidor polypeptide, and n is 0 or 1. For instance, the polypeptide chain(s)may comprise: V_(H)-C_(H)1-flexible linker-V_(H)-C_(H)1-Fc region chain;or V_(H)-C_(H)1-V_(H)-C_(H)1-Fc region chain. The multivalent antibodyherein preferably further comprises at least two (and preferably four)light chain variable domain polypeptides. The multivalent antibodyherein may, for instance, comprise from about two to about eight lightchain variable domain polypeptides. The light chain variable domainpolypeptides contemplated here comprise a light chain variable domainand, optionally, further comprise a C_(L) domain.

7) Heteroconjugate Antibodies

Heteroconjugate antibodies can also be used as the first antigen bindingportion of the MABPs of the present application. Heteroconjugateantibodies are composed of two covalently joined antibodies. Forexample, one of the antibodies in the heteroconjugate can be coupled toavidin, the other to biotin. Such antibodies have, for example, beenproposed to target immune system cells to unwanted cells, U.S. Pat. No.4,676,980, and for treatment of HIV infection. WO 91/00360, WO 92/200373and EP 0308936. It is contemplated that the antibodies may be preparedin vitro using known methods in synthetic protein chemistry, includingthose involving crosslinking agents. For example, immunotoxins may beconstructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980. Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

8) Effector Function Engineering

It may be desirable to modify the MABPs of the present application withrespect to Fc effector function, e.g., so as to modify (e.g., enhance oreliminate) antigen-dependent cell-mediated cytotoxicity (ADCC) and/orcomplement dependent cytotoxicity (CDC) of the antibody. In a preferredembodiment, Fc effector function of the MABP is reduced or eliminated.This may be achieved by introducing one or more amino acid substitutionsin an Fc region of the antibody. Alternatively or additionally, cysteineresidue(s) may be introduced in the Fc region, thereby allowinginterchain disulfide bond formation in this region. The homodimeric MABPthus generated may have improved internalization capability and/orincreased complement-mediated cell killing and antibody-dependentcellular cytotoxicity (ADCC). See Caron et al., J. Exp Med.176:1191-1195 (1992) and Shopes, B. J. Immunol. 148:2918-2922 (1992).Homodimeric antibodies with enhanced anti-tumor activity may also beprepared using heterobifunctional cross-linkers as described in Wolff etal., Cancer Research 53:2560-2565 (1993). Alternatively, an antibody canbe engineered which has dual Fc regions and may thereby have enhancedcomplement lysis and ADCC capabilities. See Stevenson et al.,Anti-Cancer Drug Design 3:219-230 (1989).

To increase the serum half-life of the antibody, one may incorporate asalvage receptor binding epitope into the MABP as described in U.S. Pat.No. 5,739,277, for example. As used herein, the term “salvage receptorbinding epitope” refers to an epitope of the Fc region of an IgGmolecule (e.g., IgG₁, IgG₂, IgG₃, or IgG₄) that is responsible forincreasing the in vivo serum half-life of the IgG molecule.

9) Other Amino Acid Sequence Modifications

Amino acid sequence modification(s) of the antibodies, such as singlechain antibodies or antibody components of the MABPs, described hereinare contemplated. For example, it may be desirable to improve thebinding affinity and/or other biological properties of the antibody.Amino acid sequence variants of the antibody are prepared by introducingappropriate nucleotide changes into the antibody nucleic acid, or bypeptide synthesis. Such modifications include, for example, deletionsfrom, and/or insertions into and/or substitutions of, residues withinthe amino acid sequences of the antibody. Any combination of deletion,insertion, and substitution is made to arrive at the final construct,provided that the final construct possesses the desired characteristics.The amino acid changes also may alter post-translational processes ofthe antibody, such as changing the number or position of glycosylationsites.

A useful method for identification of certain residues or regions of theantibody that are preferred locations for mutagenesis is called “alaninescanning mutagenesis” as described by Cunningham and Wells in Science,244:1081-1085 (1989). Here, a residue or group of target residues areidentified (e.g., charged residues such as arg, asp, his, lys, and glu)and replaced by a neutral or negatively charged amino acid (mostpreferably alanine or polyalanine) to affect the interaction of theamino acids antigen. Those amino acid locations demonstrating functionalsensitivity to the substitutions then are refined by introducing furtheror other variants at, or for, the sites of substitution. Thus, while thesite for introducing an amino acid sequence variation is predetermined,the nature of the mutation per se need not be predetermined. Forexample, to analyze the performance of a mutation at a given site, alascanning or random mutagenesis is conducted at the target codon orregion and the expressed antibody variants are screened for the desiredactivity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue or the antibody fusedto a cytotoxic polypeptide. Other insertional variants of the antibodymolecule include the fusion to the N- or C-terminus of the antibody toan enzyme (e.g. for ADEPT) or a polypeptide which increases the serumhalf-life of the antibody.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the antibody moleculereplaced by a different residue. The sites of greatest interest forsubstitutional mutagenesis include the hypervariable regions, but FRalterations are also contemplated. Conservative substitutions are shownin the Table 2 below under the heading of “preferred substitutions”. Ifsuch substitutions result in a change in biological activity, then moresubstantial changes, denominated “exemplary substitutions” in Table II,or as further described below in reference to amino acid classes, may beintroduced and the products screened.

TABLE II Amino Acid Substitutions Original Exemplary Preferred ResidueSubstitutions Substitutions Ala (A) val; leu; ile val Arg (R) lys; gln;asn lys Asn (N) gln; his; asp, lys; arg gln Asp (D) glu; asn glu Cys (C)ser; ala ser Gln (Q) asn; glu asn Glu (E) asp; gln asp Gly (G) ala alaHis (H) asn; gln; lys; arg arg Ile (I) leu; val; met; ala; phe;norleucine leu Leu (L) norleucine; ile; val; met; ala; phe ile Lys (K)arg; gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala;tyr tyr Pro (P) Ala ala Ser (S) Thr thr Thr (T) Ser ser Trp (W) tyr; phetyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile; leu; met; phe; ala;norleucine leu

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;(2) neutral hydrophilic: cys, ser, thr;(3) acidic: asp, glu;(4) basic: asn, gin, his, lys, arg;(5) residues that influence chain orientation: gly, pro; and(6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

Any cysteine residue not involved in maintaining the proper conformationof the antibody also may be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcrosslinking. Conversely, cysteine bond(s) may be added to the antibodyto improve its stability (particularly where the antibody is an antibodyfragment such as an Fv fragment).

A particularly preferred type of substitutional variant involvessubstituting one or more hypervariable region residues of a parentantibody (e.g. a humanized or human antibody). Generally, the resultingvariant(s) selected for further development will have improvedbiological properties relative to the parent antibody from which theyare generated. A convenient way for generating such substitutionalvariants involves affinity maturation using phage display. Briefly,several hypervariable region sites (e.g. 6-7 sites) are mutated togenerate all possible amino substitutions at each site. The antibodyvariants thus generated are displayed in a monovalent fashion fromfilamentous phage particles as fusions to the gene III product of M13packaged within each particle. The phage-displayed variants are thenscreened for their biological activity (e.g. binding affinity) as hereindisclosed. In order to identify candidate hypervariable region sites formodification, alanine scanning mutagenesis can be performed to identifyhypervariable region residues contributing significantly to antigenbinding. Alternatively, or additionally, it may be beneficial to analyzea crystal structure of the antigen-antibody complex to identify contactpoints between the antibody and its target (e.g., PD-L1, B7.1). Suchcontact residues and neighboring residues are candidates forsubstitution according to the techniques elaborated herein. Once suchvariants are generated, the panel of variants is subjected to screeningas described herein and antibodies with superior properties in one ormore relevant assays may be selected for further development.

Another type of amino acid variant of the antibody alters the originalglycosylation pattern of the antibody. By altering is meant deleting oneor more carbohydrate moieties found in the antibody, and/or adding oneor more glycosylation sites that are not present in the antibody.

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

Nucleic acid molecules encoding amino acid sequence variants to theMABPs of the present application are prepared by a variety of methodsknown in the art. These methods include, but are not limited to,isolation from a natural source (in the case of naturally occurringamino acid sequence variants) or preparation by oligonucleotide-mediated(or site-directed) mutagenesis, PCR mutagenesis, and cassettemutagenesis of an earlier prepared variant or a non-variant versions.

10) Other Modifications

The MABPs of the present application can be further modified to containadditional nonproteinaceous moieties that are known in the art andreadily available. Preferably, the moieties suitable for derivatizationof the antibody are water-soluble polymers. Non-limiting examples ofwater-soluble polymers include, but are not limited to, polyethyleneglycol (PEG), copolymers of ethylene glycol/propylene glycol,carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, polyaminoacids (either homopolymers or randomcopolymers), and dextran or poly(n-vinyl pyrrolidone)polyethyleneglycol, polypropylene glycol homopolymers, polypropylene oxide/ethyleneoxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, and mixtures thereof. Polyethylene glycol propionaldehyde mayhave advantages in manufacturing due to its stability in water. Thepolymer may be of any molecular weight, and may be branched orunbranched. The number of polymers attached to the antibody may vary,and if more than one polymer is attached, they can be the same ordifferent molecules. In general, the number and/or type of polymers usedfor derivatization can be determined based on considerations including,but not limited to, the particular properties or functions of theantibody to be improved, whether the antibody derivative will be used ina therapy under defined conditions, etc. Such techniques and othersuitable formulations are disclosed in Remington: The Science andPractice of Pharmacy, 20th Ed., Alfonso Gennaro, Ed., PhiladelphiaCollege of Pharmacy and Science (2000).

VI. Kits and Articles of Manufacture

Further provided are kits, unit dosages, and articles of manufacturecomprising any of the MABPs described herein. In some embodiments, a kitis provided comprising any one of the pharmaceutical compositionsdescribed herein and preferably provides instructions for its use.

The kits of the present application are in suitable packaging. Suitablepackaging includes, but is not limited to, vials, bottles, jars,flexible packaging (e.g., sealed Mylar or plastic bags), and the like.Kits may optionally provide additional components such as buffers andinterpretative information. The present application thus also providesarticles of manufacture, which include vials (such as sealed vials),bottles, jars, flexible packaging, and the like.

The article of manufacture can comprise a container and a label orpackage insert on or associated with the container. Suitable containersinclude, for example, bottles, vials, syringes, etc. The containers maybe formed from a variety of materials such as glass or plastic.Generally, the container holds a composition which is effective fortreating a disease or disorder described herein, and may have a sterileaccess port (for example the container may be an intravenous solutionbag or a vial having a stopper pierceable by a hypodermic injectionneedle). The label or package insert indicates that the composition isused for treating the particular condition in an individual. The labelor package insert will further comprise instructions for administeringthe composition to the individual. The label may indicate directions forreconstitution and/or use. The container holding the pharmaceuticalcomposition may be a multi-use vial, which allows for repeatadministrations (e.g. from 2-6 administrations) of the reconstitutedformulation. Package insert refers to instructions customarily includedin commercial packages of therapeutic products that contain informationabout the indications, usage, dosage, administration, contraindicationsand/or warnings concerning the use of such therapeutic products.Additionally, the article of manufacture may further comprise a secondcontainer comprising a pharmaceutically-acceptable buffer, such asbacteriostatic water for injection (BWFI), phosphate-buffered saline,Ringer's solution and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, and syringes.

The kits or article of manufacture may include multiple unit doses ofthe pharmaceutical composition and instructions for use, packaged inquantities sufficient for storage and use in pharmacies, for example,hospital pharmacies and compounding pharmacies.

EXAMPLES

The examples below are intended to be purely exemplary of the inventionand should therefore not be considered to limit the invention in anyway. The following examples and detailed description are offered by wayof illustration and not by way of limitation.

Example 1: Construction, Expression and Biophysical Characterization ofPD-1/CTLA-4 Bispecific Antigen Binding Proteins

This example describes the construction and expression of exemplaryPD-1/CTLA-4 bispecific antigen binding proteins (BABP). 15 constructswere designed and expressed, each comprising two polypeptide chains asfollows.

Constructs 1-3 (BCP-73, BCP-74, BCP-75): The first polypeptide comprisesfrom the N-terminus to the C terminus: the V_(H)H domain of ananti-CTLA-4 sdAb (sdAb-1 for BCP-73, sdAb-2 for BCP-74, and sdAb-3 forBCP-75), a peptide linker (a modified sequence from human IgG1 hingeregion, e.g., SEQ ID NO: 13), the heavy chain variable domain V_(H) ofpembrolizumab, and heavy chain constant domains of IgG4. The secondpolypeptide comprises from the N-terminus to the C-terminus: the lightchain variable domain V_(L) of pembrolizumab, and antibody kappa lightchain C_(L) domain. The three BABPs have the format of FIG. 9 .

Construct 4-6 (BCP-78, BCP-79, BCP-80): The first polypeptide comprisesfrom the N-terminus to the C terminus: the V_(H)H domain of ananti-CTLA-4 sdAb (sdAb-1 for BCP-78, sdAb-2 for BCP-79, and sdAb-3 forBCP-80), a peptide linker (SEQ ID NO: 13), the heavy chain variabledomain V_(H) of nivolumab, and heavy chain constant domains of IgG4. Thesecond polypeptide comprises from the N-terminus to the C-terminus: thelight chain variable domain V_(L) of nivolumab, and antibody kappa lightchain C_(L) domain. The three BABPs have the format of FIG. 9 .

Construct 7 (BCP-2): The first polypeptide comprises from the N-terminusto the C terminus: the heavy chain variable domain V_(H) ofpembrolizumab, heavy chain constant domains of IgG4, a peptide linker(GGGGSGGGS, SEQ ID NO: 1), and the V_(H)H domain of an anti-CTLA-4 sdAb(sdAb-1). The second polypeptide comprises from the N-terminus to theC-terminus: the light chain variable domain V_(L) of pembrolizumab, andantibody kappa light chain C_(L) domain. BCP-2 has the format of FIG. 4.

Construct 8 (BCP-16): The first polypeptide comprises from theN-terminus to the C terminus: the heavy chain variable domain V_(H) ofpembrolizumab, and heavy chain constant domains of IgG4. The secondpolypeptide comprises from the N-terminus to the C-terminus: the V_(H)Hdomain of an anti-CTLA-4 sdAb (sdAb-1), a peptide linker (human IgG1hinge region, e.g., SEQ ID NO: 8), the light chain variable domain V_(L)of pembrolizumab, and antibody kappa light chain C_(L) domain. BCP-16has the format of FIG. 13 .

Construct 9 (BCP-17): The first polypeptide comprises from theN-terminus to the C terminus: the heavy chain variable domain V_(H) ofpembrolizumab, and heavy chain constant domains of IgG4. The secondpolypeptide comprises from the N-terminus to the C-terminus: the lightchain variable domain V_(L) of pembrolizumab, antibody kappa light chainC_(L) domain, a peptide linker (SEQ ID NO: 8), the V_(H)H domain of ananti-CTLA-4 sdAb (sdAb-1). BCP-17 has the format of FIG. 11 .

Construct 10 (BCP-31): The first polypeptide comprises from theN-terminus to the C terminus: the V_(H)H domain of an anti-CTLA-4 sdAb(sdAb-1), a peptide linker (SEQ ID NO: 1), the heavy chain variabledomain V_(H) of pembrolizumab, and heavy chain constant domains of IgG4.The second polypeptide comprises from the N-terminus to the C-terminus:the V_(H)H domain of an anti-CTLA-4 sdAb (sdAb-1), a peptide linker (SEQID NO: 1), the light chain variable domain V_(L) of pembrolizumab, andantibody kappa light chain C_(L) domain. BCP-31 has the format of FIG.17 .

Construct 11 (BCP-32): The first polypeptide comprises from theN-terminus to the C terminus: the V_(H)H domain of an anti-CTLA-4 sdAb(sdAb-1), a peptide linker (SEQ ID NO: 1), the V_(H)H domain of ananti-CTLA-4 sdAb (sdAb-1), a peptide linker (SEQ ID NO: 1), the heavychain variable domain V_(H) of pembrolizumab, and heavy chain constantdomains of IgG4. The second polypeptide comprises from the N-terminus tothe C-terminus: the light chain variable domain V_(L) of pembrolizumab,and antibody kappa light chain C_(L) domain. BCP-32 has the format ofFIG. 18 .

Construct 12 (BCP-33): The first polypeptide comprises from theN-terminus to the C terminus: the heavy chain variable region ofpembrolizumab, constant CH1 region of IgG4, a peptide linker (SEQ ID NO:8), the V_(H)H domain of an anti-CTLA-4 sdAb (sdAb-1), and the Fc regionof IgG1. The second polypeptide comprises from the N-terminus to theC-terminus: the light chain variable domain V_(L) of pembrolizumab, andantibody kappa light chain C_(L) domain. BCP-33 has the format of FIG.19 .

Construct 13 (BCP-34): The polypeptide comprises from the N-terminus tothe C terminus: the light chain variable domain V_(L) of pembrolizumab,a peptide linker (GGGGSGGGGSGGGGS, SEQ ID NO: 12), the heavy chainvariable domain V_(H) of pembrolizumab, a peptide linker (SEQ ID NO: 8),the V_(H)H domain of an anti-CTLA-4 sdAb (sdAb-1), and Fc region ofIgG1. BCP-34 has the format of FIG. 20 .

Construct 14 (BCP-35): The first polypeptide comprises from theN-terminus to the C terminus: the heavy chain variable region ofpembrolizumab, constant CH1 region of IgG4, a peptide linker (SEQ ID NO:8), the V_(H)H domain of an anti-CTLA-4 sdAb (sdAb-1), constant CH1region of IgG4, and the Fc region of IgG4. The second polypeptidecomprises from the N-terminus to the C-terminus: the light chainvariable region of pembrolizumab, antibody kappa light chain C_(L)domain, a peptide linker (SEQ ID NO: 8), the V_(H)H domain of ananti-CTLA-4 sdAb (sdAb-1), and antibody kappa light chain C_(L) domain.BCP-35 has the format of FIG. 21 .

Construct 15 (BCP-36): The first polypeptide comprises from theN-terminus to the C terminus: the light chain variable domain V_(L) ofpembrolizumab, a peptide linker (SEQ ID NO: 12), the heavy chainvariable domain V_(H) of pembrolizumab, a peptide linker (SEQ ID NO: 8),the V_(H)H domain of an anti-CTLA-4 sdAb (sdAb-1), and Fc region ofIgG1. The second polypeptide comprises from the N-terminus to theC-terminus: the V_(H)H domain of an anti-CTLA-4 sdAb (sdAb-1), andantibody kappa light chain C_(L) domain. BCP-36 has the format of FIG.22 .

Each BABP consists of two copies of the first polypeptide and two copiesof the second polypeptide. An S228P mutation can be introduced to theIgG4 Fc region to inhibit Fab arm exchange. Furthermore, the Fc regionof the BABP may be swapped with an IgG Fc of a different isotype, forexample, the IgG1 isotype. The Fc region of IgG4 isotype has low bindingaffinity to FcγRs, and thus is preferable over IgG1 isotype in someembodiments for avoiding ADCC-mediated depletion of PD-1 or CTLA-4positive cells.

Production

The plasmids of the 15 BABP constructs described above were prepared andtransiently expressed in CHO-3E7 cells. The BABPs were purified byone-step protein A chromatography and stored in PBS buffer, pH7.4. Thecomposition and purity of the purified BABPs were analyzed by SDS-PAGEunder both reduced and non-reduced conditions. The sizes of thepolypeptide chains as well as the full-length BABP molecules wereconsistent with their calculated molecular mass based on the amino acidsequences. To further study the physical properties of the BABPs insolution, size exclusion chromatography was used to analyze eachprotein. All BABPs exhibited a single major peak, demonstrating physicalhomogeneity as monomeric molecules, i.e., non-aggregated BABP moleculeseach being a dimeric protein consisting of 4 polypeptide chains,including 2 copies of the first polypeptide chain and 2 copies of thesecond polypeptide chain. A summary of this data is shown in theTable 1. Data in Table 1 shows that the production levels of most BABPsare comparable to those of the regular monoclonal antibodies, indicatingthat the BABPs can be expressed efficiently in mammalian cells.

The purified BABPs could also be formulated in a solution containingsodium citrate, sodium chloride, mannitol, diethylenetriaminepentaceticacid (pentetic acid), and polysorbate 80 (Tween 80), pH 6.0.

TABLE 1 Production of exemplary PD-1/CTLA-4 BABPs. Transient Monomericexpression molecule Storage BABP Host cell (mg/L) (HPLC) buffer BCP-73CHO-3E7 13.15 94.20% PBS, pH 7.2 BCP-74 CHO-3E7 13.45 94.00% PBS, pH 7.2BCP-75 CHO-3E7 14.55 94.70% PBS, pH 7.2 BCP-78 CHO-3E7 106.3 96.60% PBS,pH 7.2 BCP-79 CHO-3E7 122.4 93.60% PBS, pH 7.2 BCP-80 CHO-3E7 102.494.10% PBS, pH 7.2 BCP-2 CHO-3E7 20.5 97.90% PBS, pH 7.2 BCP-16 CHO-3E74.65 98.30% PBS, pH 7.2 BCP-17 CHO-3E7 12.35 92.30% PBS, pH 7.2 BCP-31CHO-3E7 31.05 95.20% PBS, pH 7.2 BCP-32 CHO-3E7 29.7 93.90% PBS, pH 7.2BCP-33 CHO-3E7 2.45 94.80% PBS, pH 7.2 BCP-34 CHO-3E7 3.6 99.50% PBS, pH7.2 BCP-35 CHO-3E7 11.25 95.40% PBS, pH 7.2 BCP-36 CHO-3E7 0.45 92.90%PBS, pH 7.2

Stability Analysis

The thermal stability of various BABPs were investigated using aMICROCAL™ VP-Capillary Differential Scanning calorimetry (DSC, Microcal,Northampton, Mass., USA, Malvern Instruments). Approximately 370 μl ofeach BABP (1 mg/ml) and its corresponding buffer was added to a 96-wellplate according to MICROCAL™ VP-Capillary DSC user's manual. A detergentcleaning program was included between each sample run to keep thereference and sample cells clean. All samples were scanned from 20° C.to 100° C. with a scan rate of 90° C./h (1.5° C./min) in a passive mode.The collected data were analyzed using the VP-Capillary DSC softwarebased on ORGIN™ 7.0 (Northampton, Mass., USA). All thermograms werecontrol and baseline subtracted to obtain the apparent midpoint (T_(m))and apparent enthalpy (ΔH) of protein unfolding. The unfolding MidpointTemperatures (T_(m)) of various BABPs are shown in Table 2 (DSC).

The formation of larger protein aggregates during heating was followedusing dynamic light scattering (DLS). A temperature ramp from 25° C. to75° C. with temperature interval at about 0.75° C. was performed forsamples at 1.5 mg/ml using the DYNAPRO® NANOSTAR® plate reader (Wyatt,Santa Barbara, Calif.). 20 μl of each BABP sample was added to a WYATT®disposable cuvette followed by covering the sample with 10 μl of mineraloil (Sigma 8410) to prevent evaporation. Triplicate measurements (5acquisitions/each measurement) were averaged for each BABP sample. Inthe duration of an experiment with the chosen temperature interval, thethermal scan rate was calculated to be 1.5° C./min. Each sample wasmeasured while the temperature was continuously heated until the targettemperature reached 75° C. (˜40 min). The aggregation temperature(T_(agg)) was analyzed with onset analysis method in the DYNAMICS™7.6.0.48 software (Wyatt, Santa Barbara, Calif.). The measuredaggregation onset temperatures (T_(agg)) of various BABPs are shown inTable 2.

TABLE 2 DSC and DLS analysis of exemplary PD-1/CTLA-4 BABPs. ConstructT_(m) (° C.) T_(agg) (° C.) BCP-73 69.5 69.2 BCP-74 68.9 70.8 BCP-7567.6 70.2 Biosimilar pembrolizumab 67.6 69.6 BCP-78 68.9 70.8 BCP-7967.9 70.6 BCP-80 67.8 69.2 Biosimilar nivolumab 65.2 67.6

BABP samples at concentration of >50 mg/ml in Histidine buffer (pH6.0)were incubated at constant temperatures of 4° C., 25° C. and 37° C. for7 days. A similar set of samples was also freeze-thawed five times.Fractions of intact full monomeric molecules of all samples wereevaluated by SEC-HPLC, and the data was recorded and analyzed usingCHROMELEON™ software supplied by the manufacturer. Table 3 shows thatthe BABPs retained greater than 90% integrity under thethermo-challenged conditions.

TABLE 3 Stability analysis of exemplary PD-1/CTLA-4 BABPs. Monomericmolecule (by SEC-HPLC) after 5 freeze- Construct Starting 4° C. 25° C.37° C. thaw cycles BCP-73 94.2% 94.8% 94.5% 93.7% 92.3% BCP-74 94.0%94.2% 93.9% 93.8% 92.5% BCP-75 94.7% 95.1% 94.5% 94.1% 93.4% BCP-7896.6% 97.2% 95.8% 95.2% 94.7% BCP-79 93.6% 94.3% 93.6% 93.1% 92.1%BCP-80 94.1% 92.8% 93.5% 92.7% 91.8%

Solubility Analysis

To characterize the solubility of purified BABPs, 10 mg of each BABP at1 mg/ml was added to MICROCON®-30 kDa centrifugal concentrators (EMDMillipore) in volumes of ˜2.5 ml and centrifuged at 4000×g (4° C.). Thevolumes were periodically checked and protein was added to theconcentrators until the remaining protein solutions had been consumed.Concentration proceeded for 2 h until either the volume reached ˜20 μlor stopped decreasing. The concentration was determined by performing UVmeasurements of samples obtained by diluting 1 μl of concentrated BABPinto 199 μl of each respective buffer. The samples were evaluated foraggregation using analytical SEC-HPLC after diluting BABPs to 1 mg/ml intheir respective buffers. Table 4 shows that the BABPs retained fullintegrity under these stressed conditions.

The solubility of purified BABPs was also measured using across-interaction chromatography (CIC) column. Murine polyclonalantibodies purified from pooled mouse serum were purchased fromSigma-Aldrich (15381). Murine polyclonal antibodies were coupled to theresin matrix at ˜30 mg/mL. Purified BABPs in PBS buffer were injected tothe murine IgG-coupled column and the control column, respectively, withconcentrations ranging from 0.05 to 0.20 mg/mL. The retention times wereused to calculate the retention factor k′ values reported in Table 4:k′=(Vr−Vo)/Vo=(Tr−Tm)/Tm. Vr represents the elution volume of the sampleon the protein coupled column, Vo represents the elution volume from acontrol column, Tr represents the retention time on the protein coupledcolumn, and Tm represents the retention time on the control column. Anumber of samples were run twice on the same column. Antibodies with k′values >0.6 are generally significantly less soluble. According to Table4, all the BABPs exhibited excellent solubility.

TABLE 4 Solubility analysis of exemplary PD-1/CTLA-4 BABPs. ConstructConcentration (mg/mL) Monemeric molecule K′ BCP-73 194.4 94.1% 0.07BCP-74 189.2 92.7% 0.04 BCP-75 290.9 92.6% 0.03 BCP-78 248.0 93.4% 0.06BCP-79 337.5 93.5% 0.04 BCP-80 206.1 92.8% 0.03

Example 2: In Vitro Functional Assays of PD-1/CTLA-4 Bispecific AntigenBinding Proteins

The 15 exemplary PD-1/CTLA-4 bispecific antigen binding proteins (BABPs)described in Example 1 were tested in the in vitro assays below toassess the functional blockade of PD-1 and CTLA-4 by the BABPs.

Target Binding Assays

The ability of the BABPs to bind PD-1 and CTLA-4 can be determined usingthe Surface Plasmon Resonance method (e.g., BIACORE®), an enzyme-linkedimmunosorbent assay, a Fluorescence-Assisted Cell Sorting method (FACS),or a combination thereof. The analyses can be performed on activated Tcells.

Binding affinities of the various BABPs to PD-1 expressed on CHO cells,were determined using a fluorescence-activated cell sorting (FACS)-basedassay. BABP samples were prepared (starting at 1 μM, 3-fold serialdilution with 10 concentrations) as primary antibodies for FACSanalysis. CHO cells expressing human PD-1 were dissociated from adherentculture flasks and mixed with varying concentrations of BABP samples(both in a 96-well plate). Pembrolizumab (e.g., KEYTRUIDA®) or nivolumab(e.g., OPDIVO®) was used as an anti-PD-1 antibody positive control. Themixture was equilibrated for 30 minutes at room temperature, washedthree times with FACS buffer (PBS containing 1% BSA). Fluoresceinisothiocyanate (FITC)-conjugated anti-human kappa antibody (JacksonImmunoResearch) used as the secondary antibody was then added andincubated for 15 minutes at room temperature. Cells were washed againwith FACS buffer and analyzed by flow cytometry. Data was analyzed withPRISM™ (GraphPad Software, San Diego, Calif.) using non-linearregression, and EC₅₀ values were calculated. As shown in Table 5, theFACS binding assays demonstrated that the BABPs retained comparable PD-1binding affinities as pembrolizumab (e.g., KEYTRUDA®) and nivolumab(e.g., OPDIVO®), respectively.

Binding affinities of the 15 BABPs to CTLA-4 expressed on CHO cells,were determined using a fluorescence-activated cell sorting (FACS)-basedassay. BABP samples were prepared (starting at 1 μM, 3-fold serialdilution with 10 concentrations) as primary antibody for FACS analysis.CHO cells expressing human CTLA-4 were dissociated from adherent cultureflasks and mixed with varying concentrations of antibodies (both in a96-well plate). sdAb-1-Fc, sdAb-2-Fc, sdAb-3-Fc and ipilimumab (e.g.,YERVOY®) were used as anti-CTLA-4 antibody positive controls. Themixture was equilibrated for 30 minutes at room temperature, washedthree times with FACS buffer (PBS containing 1% BSA). Fluoresceinisothiocyanate (FITC)-conjugated anti-human kappa antibody (JacksonImmunoResearch) used as the secondary antibody was then added andincubated for 15 minutes at room temperature. Cells were washed againwith FACS buffer and analyzed by flow cytometry. Data were analyzed withPRISM™ (GraphPad Software, San Diego, Calif.) using non-linearregression, and EC₅₀ values were calculated. As show in Table 5, theFACS binding assays demonstrated that the BABPs exhibited comparablebinding affinities to CTLA-4 as their corresponding sdAbs fused to anFc. Also, the BABPs showed comparable binding affinities to CTLA-4 asipilimumab (e.g., YERVOY®).

Binding kinetics of various BABPs to PD-1 were determined using aSurface Plasmon Resonance (SPR) biosensor, BIACORE® T200 (GEHealthcare). Different concentrations of the BABP samples were preparedstarting at 50 nM with 3-fold serial dilution. Each BABP sample wasimmobilized on the sensor chip through the Fc capture method. AntigenPD-1 was used as the analyte. The dissociation (k_(d)) and association(k_(a)) rate constants were obtained using the BIACORE® T200 evaluationsoftware. The apparent equilibrium dissociation constants (K_(D)) werecalculated from the ratio of k_(d) over k_(a). As shown in Table 5, theBABPs retained comparable binding kinetics to PD-1 as pembrolizumab(e.g., KEYTRUDA®) and nivolumab (e.g., OPDIVO®).

Binding kinetics of various BABPs to CTLA-4 were determined using aSurface Plasmon Resonance (SPR) biosensor, BIACORE® T200 (GEHealthcare). Different concentrations of the BABP samples were preparedstarting at 200 nM with 3-fold serial dilution. Each BABP sample wasimmobilized on the sensor chip through the Fc capture method. AntigenCTLA-4 was used as the analyte. The dissociation (k_(d)) and association(k_(a)) rate constants were obtained using the BIACORE® T200 evaluationsoftware. The apparent equilibrium dissociation constants (K_(D)) werecalculated from the ratio of k_(d) over k_(a). As shown in Table 5, thebinding kinetics demonstrated that the BABPs exhibited comparablebinding kinetics to CTLA-4 as their corresponding sdAbs fused to an Fc.Also, the BABPs have comparable binding kinetics to CTLA-4 as biosimilaripilimumab.

TABLE 5 Binding data of exemplary PD-1/CTLA-4 BABPs. PD-1 CTLA-4 K_(D)EC₅₀ IC₅₀ K_(D) EC₅₀ IC₅₀ Construct (nM) (nM) (nM) (nM) (nM) (nM) BCP-737.5 2.2 1.1 7.5 3.0 11.5 BCP-74 2.5 1.6 2.6 2.6 3.9 17.2 BCP-75 2.2 1.61.5 4.0 3.0 11.7 BCP-78 8.1 1.7 4.8 6.6 2.7 7.1 BCP-79 6.8 1.1 3.5 2.51.9 7.8 BCP-80 6.3 1.4 5.7 5.6 3.7 11.9 BCP-2 5.3 5.2 2.3 11.0 16.4 11.7BCP-16 3.9 12.2 8.8 4.8 26.6 8.6 BCP-17 3.9 2.7 4.0 39.3 17.7 33.3BCP-31 8.0 3.4 5.7 4.3 31.2 15.3 BCP-32 7.5 8.1 4.3 4.1 71.7 14.6 BCP-338.1 1.5 2.0 9.2 48.0 26.3 BCP-34 9.2 5.4 4.8 6.3 24.3 18.6 BCP-35 7.33.0 4.6 7.2 20.4 17.5 BCP-36 8.3 1.8 2.0 6.1 26.4 17.9 pembrolizumab 6.51.1 1.3 N/A N/A N/A (KEYTRUDA ®) nivolumab 7.3 1.1 3.1 N/A N/A N/A(OPDIVO ®) sdAb-1 N/A N/A N/A 15.0 2.1 3.5 sdAb-2 N/A N/A N/A 4.2 3.24.1 sdAb-3 N/A N/A N/A 5.5 3.5 5.1 ipilimumab N/A N/A N/A 17.3 13.2 8.5(YERVOY ®)

Inhibition of Ligand Binding by FACS Analysis

Inhibition of ligand binding by the BABPs was assessed using a FACSassay.

To assess inhibition of PD-L1 by the BABPs, BABP samples were prepared(starting at 1 μM, 3-fold serial dilution with 10 concentrations). CHOcells expressing human PD-1 were dissociated from adherent cultureflasks and mixed with varying concentrations of each BABP and 0.5 μMhPD-L1-Fc fusion protein having a biotin label. Biosimilar pembrolizumabor biosimilar nivolumab was used as an anti-PD-1 antibody positivecontrol. The mixture was equilibrated for 30 minutes at roomtemperature, and washed three times with FACS buffer (PBS containing 1%BSA). PE/Cy5 Streptavidin secondary antibody was then added to themixtures and incubated for 15 minutes at room temperature. Subsequently,the cells were washed with FACS buffer and analyzed by flow cytometry.Data was analyzed with PRISM™ (GraphPad Software, San Diego, Calif.)using non-linear regression, and IC₅₀ values were calculated (Table 5).The competition assays demonstrated the ability of the BABPs toefficiently inhibit PD-1/PD-L1 interactions at low concentrations (1-10μg/ml). The binding data in Table 5 indicates that the functionalactivities of the exemplary PD1/CTLA-4 BABPs are very similar topembrolizumab (e.g., KEYTRUDA®) and nivolumab (e.g., OPDIVO®).

To assess inhibition of B7-1 (a CTLA-4 ligand) by the BABPs, BABPsamples were prepared (starting at 1 μM, 3-fold serial dilution with 10concentrations). CHO cells expressing human B7-1 cells were dissociatedfrom adherent culture flasks and mixed with varying concentrations ofeach BABP and 0.5 μM hCTLA-4-Fc fusion protein having a biotin-label.sdAb-1-Fc, sdAb-2-Fc, sdAb-3-Fc and ipilimumab (e.g., YERVOY®) were usedas anti-CTLA-4 antibody positive controls. The mixture was equilibratedfor 30 minutes at room temperature, and washed three times with FACSbuffer (PBS containing 1% BSA). PE/Cy5 Streptavidin secondary antibodywas then added to the mixtures and incubated for 15 minutes at roomtemperature. Subsequently, the cells were washed again with FACS bufferand analyzed by flow cytometry. Data were analyzed with PRISM™ (GraphPadSoftware, San Diego, Calif.) using non-linear regression, and IC₅₀values were calculated (Table 5). The competition assays demonstratedthe ability of the BABPs to efficiently inhibit CTLA4-B7-1 interactionsat low concentrations (1-10 μg/ml). The binding data in Table 5indicates that the functional activities of the exemplary PD1/CTLA-4BABPs are similar to their corresponding sdAbs fused to an Fc andbiosimilar ipilimumab.

The expression profile and dual-binding properties of the BABPs clearlydemonstrate bispecificity of the BABPs, which have a first specificityprovided by the antigen binding site formed by correct pairing of theV_(H) and V_(L) of the 4-chain antibody, and the second specificityprovided by the V_(H)Hs.

In Vitro Functional Assays

Blockade of the PD-1 and CTLA-4 pathways by the BABPs can be studiedusing a variety of bioassays that monitor T cell proliferation, IFN-γrelease, IL-2 secretion or expression of reporter gene that is driven bysignaling in the PD-1 or CTLA-4 pathway.

The BCP-73, BCP-74, BCP-75, BCP-78, BCP-79 and BCP-80 6 BABPs wereselected for in vitro bioactivity evaluation. Characterization ofbiological activity of anti-PD-1 neutralizing antibody in PD-1/PD-L1cell-based assay using the PD-1/NFAT Reporter-Jurkat cells is shown inTable 6. In this case, CHO-K1 cells were stably expressed with humanPD-L1 and an engineered T cell receptor (TCR) activator. The affectercells-PD-1/NFAT Reporter-Jurkat cells were pre-incubated with serialdilution of BABPs for 30 minutes prior to co-culture with engineeredCHO-K1 cells. After ˜6 hours of stimulation, ONE-STEP™ Luciferasereagent was added to the cells to measure NFAT activity. Data wasanalyzed with PRISM™ (GraphPad Software, San Diego, Calif.) usingnon-linear regression, and EC₅₀ values were calculated. The reporterassay demonstrated the ability of all BABPs to efficiently activate NFATsignal similar as pembrolizumab (e.g., KEYTRUDA) and nivolumab (e.g.,OPDIVO®).

TABLE 6 Antibody biological activity PD-1 CTLA-4 Construct EC₅₀ (nM)EC₅₀ (nM) BCP-73 1.6 12.1 BCP-74 4.3 14.3 BCP-75 5.5 12.1 BCP-78 8.9 8.4BCP-79 6.5 10.9 BCP-80 4.7 7.4 pembrolizumab (KEYTRUDA ®) 1.5 N/Anivolumab (OPDIVO ®) 3.3 N/A sdAb-1-Fc N/A 12.1 sdAb-2-Fc N/A 12.9sdAb-3-Fc N/A 13.1 ipilimumab (YERVOY ®) N/A 17.6

The bispecific antibodies are found to effectively inhibit the bindingbetween CTLA-4 and B7-1 as shown in Table 6 using CTLA-4 cell-basedblockade assay. Briefly, human CD4⁺ T cells were purified from PBMC bythe isolation kits (Miltenyl Biotec). Each well contained 10⁵ CD4⁺ Tcells and 10⁴ CHO-K1/human CD80 (CHO-K1 stably expressing human CD80)with a final working volume of 200 μl. Bispecific antibodies were addedinto each well at different concentrations. No antibody was used as abackground control. Human IgG4 was used as a negative control, andipilimumab (e.g., YERVOY®) was used as a positive anti-CTLA4 antibodycontrol. CTLA-4-Fc (GenScript, Z03373-50) was added into the system toinitiate the reaction. After 24-hour incubation in 37° C./5% CO₂incubator, 100 μl medium was taken from each testing well for IL-2measurement (Cisbio). Antibody concentration-dependent secretion of IL-2by T cells in the CTLA-4 blockade bioassays was used to extract an EC₅₀value for each test antibody, as well as for the positive controlfull-length anti-CTLA-4 antibody ipilimumab (e.g., YERVOY®).

PD-1 pathway inhibition by the BCP-74, BCP-75, BCP-79 and BCP-80 BABPswere studied by determining the IL-2 and IFN-γ secretion level in mixedlymphocyte reactions (MLR) containing target cells expressing PD-L1(such as dendritic cells), activated T cells, and each of the BABPs.Human CD4⁺ T cells and allogeneic monocytes are purified from PBMC usingisolation kits (Miltenyl Biotec). Monocytes were induced into dendriticcells. Each well contains 10⁵ CD4⁺ T cells and 10⁴ allogeneic dendriticcells with a final working volume of 200 μl. Each of the BABPs was addedinto each well at different concentrations. A no antibody well was usedas the background control. Human IgG4 was used as the negative controland pembrolizumab (e.g., KEYTRUDA®) was used as the positive anti-PD-1antibody control. After incubating for 72 hours at 37° C. in a 5% CO₂incubator, 100 μl medium was taken from each testing well for IL-2 andIFN-γ measurement (Cisbio). Concentration-dependent secretion of IL-2and IFN-γ in the MLRs is used to extract an EC₅₀ value for the BABPsagainst PD-1, which is compared with the EC₅₀ value of control PD-1antibody pembrolizumab (e.g., KEYTRUDA®). As shown in Table 7, variousBABPs exhibit comparable inhibition potential to pembrolizumab (e.g.,KEYTRUDA®).

TABLE 7 Mixed lymphocyte reactions of PD-1/CTLA-4 BABPs Construct IFN-γEC50 (nM) IL-2 EC50 (nM) BCP-74 1.24 1.15 BCP-75 0.51 2.1 pembrolizumab(KEYTRUDA ®) 0.92 1.68 BCP-79 0.50 2.33 BCP-80 0.30 1.00 nivolumab(OPDIVO ®) 1.77 1.76

Example 3: In Vivo Anti-Tumor Efficacy of PD-1/CTLA-4 Bispecific AntigenBinding Proteins

This example describes in vivo experiments assessing the functionalblockade of PD-1 and CTLA-4 by the BCP-75 and BCP-79 BABPs. Anti-tumorefficacy was evaluated in tumor models developed with human CTLA-4 andPD-1 Knock-in mice. Humanization of both CTLA-4 and PD-1 in mice enableddirect in vivo evaluation of the efficacy of PD-1/CTLA-4 BABPs in amouse tumor xenograft model.

The mouse xenograft models can be prepared by implanting tumor cellsinto NSG mice. Tumor cell lines, such as MC38 (a murine colonadenocarcinoma cell line) and CT26 (a murine colon carcinoma cell line),can be used to prepare mouse models for colon cancer. B16, a murinemelanoma cell line, can be used to prepare a mouse model for melanoma.Renca, a murine renal cortical adenocarcinoma cell line, can be used toprepare a mouse model for renal cancer.

6-8-week-old human PD-1 KI female C57/BL6 mice were shaved on theirlower dorsum and s.c. injected with 1×10⁶ colon cancer cell line MC38 ina 50 μL suspension of 75% (vol/vol) RPMI (Life Technologies) and 25%(vol/vol) medium-density MATRIGEL® (Corning). Mice whose tumors failedto engraft within 7 days by visual inspection were excluded from furtherstudy. Tumors were measured on a daily basis starting at day 7 afterMC38 engraftment. Mice were individually sorted into treatment cohorts,and started to receive treatment only when tumors reached a threshold of150 mm³, about 10 days post engraftment in all cases. Digital calipermeasurements and body weight measurements were taken every three daysfor the duration of treatment. In the experiments, mice were giventreatment intravenously for 16 days with 10 mg/kg biosimilarpembrolizumab, 10 mg/kg biosimilar nivolumab, or 12.3 mg/kg BABP (BCP-75or BCP-79). The treatment was administered every 4 days. As shown inFIG. 23 , both BCP-75 and BCP-79 effectively controlled tumor growth inthe MC38 syngeneic mice model, exhibiting comparable functionalactivities as biosimilar pembrolizumab and biosimilar nivolumab. None ofthe three treatment regimens affected the body weights of the MC38engrafted mice, as compared to the mock control (data not shown).

6-8-week-old human CTLA-4 KI female C57/BL6 mice were shaved on theirlower dorsum and s.c. injected with 1×10⁶ colon cancer cell line MC38 ina 50 μl suspension of 75% (vol/vol) RPMI (Life Technologies) and 25%(vol/vol) medium-density MATRIGEL® (Corning). Mice whose tumors failedto engraft within 7 days by visual inspection were excluded from furtherstudy. Tumors were measured on a daily basis starting at day 7 afterMC38 engraftment. Mice were individually sorted into treatment cohorts,and started to receive treatment only when tumors reached a threshold of150 mm³, about 10 days post engraftment in all cases. Digital calipermeasurements and body weight measurements were taken every three daysfor the duration of treatment. In the experiments, mice were giventreatment intravenously for 16 days with 10 mg/kg biosimilar ipilimumab,12.3 mg/kg BABP (BCP-75 or BCP-79), or 6.7 mg/kg of sdAb-2-Fc orsdAb-3-Fc. The treatment was administered every 4 days. As shown in FIG.24 , both BCP-75 and BCP-79 effectively controlled tumor growth in theMC38 syngeneic mice model, exhibiting comparable functional activitiesas sdAb-2-Fc and sdAb-3-Fc. None of the three treatment regimensaffected the body weights of the MC38 engrafted mice, as compared to themock control (data not shown).

Example 4: Construction, Expression and Biophysical Characterization ofPD-L1/CTLA-4 Bispecific Antigen Binding Proteins

This example describes the construction and expression of exemplaryPD-L1/CTLA-4 BABPs. Two constructs were designed and expressed, eachcomprising two polypeptide chains as follows:

BCP-84: The first polypeptide comprises from the N-terminus to the Cterminus: the V_(H)H domain of an anti-CTLA-4 sdAb-2, a peptide linker(SEQ ID NO: 13), the heavy chain variable domain V_(H) of atezolizumab,and heavy chain constant domains of non-glycosylated IgG1. The secondpolypeptide comprises from the N-terminus to the C-terminus: the lightchain variable domain V_(L) of atezolizumab, and antibody kappa lightchain C_(L) domain. BCP-84 has the format of FIG. 9 .

BCP-85: The first polypeptide comprises from the N-terminus to the Cterminus: the V_(H)H domain of an anti-CTLA-4 sdAb-3, a peptide linker(SEQ ID NO: 13), the heavy chain variable domain V_(H) of atezolizumab,and heavy chain constant domain of non-glycosylated IgG1. The secondpolypeptide comprises from the N-terminus to the C-terminus: the lightchain variable domain V_(L) of atezolizumab, and antibody kappa lightchain C_(L) domain. BCP-85 has the format of FIG. 9 .

BCP-84 and BCP-85 each consists of two copies of the first polypeptideand two copies of the second polypeptide. The IgG1 Fc region for theconstructs was a non-glycosylated IgG1. Furthermore, the Fc region ofthe bispecific antigen binding protein may be swapped with IgG Fc of adifferent isotype, for example, the wild-type IgG1 isotype for the IgG4isotype with S228P mutation. The Fc region of non-glycosylated IgGisotype has no binding affinity to FcγRs, and thus is preferable overwild-type IgG1 isotype in some embodiments for avoiding ADCC-mediateddepletion of PD-L1 or CTLA-4 positive cells.

Production

The plasmids of BCP-84 and BCP-85 were prepared and transientlyexpressed in CHO cells. The BABPs were purified by one-step protein Achromatography and stored in PBS buffer, pH7.4. The composition andpurity of the purified BABPs were analyzed by SDS-PAGE under bothreduced and non-reduced conditions. The sizes of the polypeptide chainsas well as the full-length protein of BABP molecules were consistentwith their calculated molecular mass based on the amino acid sequences.To further study the physical properties of the 2 BABPs in solution,size exclusion chromatography was used to analyze each protein. BothBABPs exhibited a single major peak, demonstrating physical homogeneityas monomeric BABP molecules. A summary of this data is shown in theTable 8.

TABLE 8 Production of exemplary PD-L1/CTLA-4 BABPs. Transient Monomericexpression molecule Storage BABP Host cell (mg/ml) (HPLC) buffer BCP-84CHO-3E7 74.4 95.34% PBS, pH 7.2 BCP-85 CHO-3E7 77.4 96.94% PBS, pH 7.2

Stability Analysis

To determine thermal stability and aggregation of the BABPs, DSC(Differential Scanning calorimetry) and DLS (Dynamic Light Scattering)experiments were carried out as described in Example 1. As shown inTable 9, Tm and Tagg of BCP-84 and BCP-85 are comparable to those ofbiosimilar atezolizumab (e.g., compared to TECENTRIQ®).

TABLE 9 DSC and DLS analysis of exemplary PD-L1/CTLA-4 BABPs. BABP T_(m)(° C.) T_(agg) (° C.) BCP-84 70.8 70.3 BCP-85 70.3 69.6 Biosimilaratezolizumab 71.8 69.2

Example 5: In Vitro Functional Assays of PD-L1/CTLA-4 Bispecific AntigenBinding Proteins

BCP-84 and BCP-85 were tested in the in vitro assays described below toassess the functional blockade of PD-L1 and CTLA-4 by the BABPs.

Target Binding Assays

The ability of the BABPs to bind PD-L1 and CTLA-4 can be determinedusing Surface Plasmon Resonance method (e.g., BIACORE®), anenzyme-linked immunosorbent assay, a Fluorescence-Assisted Cell Sortingmethod (FACS), or a combination thereof. The analyses can be performedon activated T cells.

Binding of each BABP to PD-L1 and CTLA-4 expressed on PD-L1 and CTLA-4expression stable cell lines, was determined using afluorescence-activated cell sorting (FACS)-based assay. BABP sampleswere prepared (starting at 1 μM, 3-fold serial dilution with 10concentrations) and incubated with PD-L1 and CTLA-4 cells. Cells boundto BCP-84 and BCP-85 BABPs were detected by an Alexa Fluor488-conjugated anti-human antibody (Jackson ImmunoResearch). The EC₅₀was calculated by GraphPad PRISM™ Version 6.0.

Binding kinetics of BCP-84 and BCP-85 to PD-L1 was determined usingHis-tagged human PD-L1 protein captured on a CMS sensor chip (Biacore).6 different samples of each BABP were prepared starting at 50 nM with3-fold serial dilution. Each BABP sample was flowed over theantigen-coated chip, and avidity was determined using Surface PlasmonResonance.

Binding kinetics of BCP-84 and BCP-85 to CTLA-4 were determined usingHis-tagged human CTLA-4 coated on a CMS sensor chip (Biacore). 6different samples of each BABP were prepared starting at 200 nM with2-fold serial dilution. Each BABP sample was flowed over theantigen-coated chip, and avidity was determined using Surface PlasmonResonance.

The affinity data of BCP-84 and BCP-85 to PD-L1 and CTLA-4 are shown inTable 10.

Inhibition of Ligand Binding by FACS Analysis

Inhibition of ligand binding by BCP-84 and BCP-85 were assessed by aFACS assay.

To assess inhibition of PD-L1 by the BABPs, CHO cells expressing humanPD-L1 were dissociated from adherent culture flasks and mixed withvarying concentrations of each BABP (starting at 1 μM, with 3-foldserial dilution for 10 concentrations) and 0.1 μM of hPD-1-Fc fusionprotein having a biotin label. The mixture was equilibrated for 30minutes at room temperature, and washed three times with FACS buffer(PBS containing 1% BSA). PE/Cy5 Streptavidin secondary antibody was thenadded to the mixtures and incubated for 15 minutes at room temperature.Subsequently, the cells were washed with FACS buffer and analyzed byflow cytometry. Data was analyzed with PRISM™ (GraphPad Software, SanDiego, Calif.) using non-linear regression, and IC50 values werecalculated and shown in Table 10. The competition assays demonstrate theability of BCP-84 and BCP-85 to efficiently inhibit PD-1/PD-L1interactions similar to biosimilar atezolizumab.

To assess inhibition of B7-1 (a CTLA-4 ligand) by BCP-84 and BCP-85, CHOcells expressing human B7-1 cells were dissociated from adherent cultureflasks and mixed with varying concentrations of each BABP (starting at 1μM, with 3-fold serial dilution for 10 concentrations) and 0.1 μM ofhCTLA-4-Fc fusion protein having a biotin label. The mixture wasequilibrated for 30 minutes at room temperature, and washed three timeswith FACS buffer (PBS containing 1% BSA). PE/Cy5 Streptavidin secondaryantibody was then added to the mixtures and incubated for 15 minutes atroom temperature. Subsequently, the cells were washed again with FACSbuffer and analyzed by flow cytometry. Data were analyzed with PRISM™(GraphPad Software, San Diego, Calif.) using non-linear regression, andIC50 values were calculated and shown in Table 10. The competitionassays demonstrate the ability of the BCP-84 and BCP-85 to efficientlyinhibit CTLA-4/B7-1 interactions similar to the corresponding sdAb-Fcand ipilimumab (e.g., YERVOY®).

TABLE 10 Binding data of exemplary PD-L1/CTLA-4 BABPs. PD-L1 CTLA-4 KDEC₅₀ IC50 KD EC₅₀ IC50 Construct (nM) (nM) (nM) (nM) (nM) (nM) BCP-840.6 3.1 2.4 4.1 2 5.5 BCP-75 0.4 3.4 2.0 3.6 3.7 5.1 Biosimilar 0.4 2.81.8 N/A N/A N/A atezolizumab sdAb-2-Fc N/A N/A N/A 4.2 3.2 4.1 sdAb-3-FcN/A N/A N/A 5.5 3.5 5.1 ipilimumab N/A N/A N/A 14.8  13.2 8.5 (YERVOY ®)

In Vitro Functional Assays

Blockade of the PD-L1 and CTLA-4 pathways by BCP-84 and BCP-85 can bestudied using a variety of bioassays that monitor T cell proliferation,IFN-γ release, IL-2 secretion or expression of reporter gene that isdriven by signaling in the PD-1 or CTLA-4 pathway.

Table 11 shows data on biological activities of anti-PD-1 neutralizingantibody in a PD-1/PD-L1 cell-based assay using the PD-1/NFATReporter-Jurkat cells. Briefly, CHO-K1 cells were stably expressed withhuman PD-L1 and an engineered T cell receptor (TCR) activator. Theaffecter cells-PD-1/NFAT Reporter-Jurkat cells were pre-incubated withserial dilution of BCP-84 and BCP-85 for 30 minutes prior to co-culturewith engineered CHO-K1 cells. After ˜6 hours of stimulation, ONE-STEP™Luciferase reagent was added to the cells to measure NFAT activity. Datawas analyzed with PRISM™ (GraphPad Software, San Diego, Calif.) usingnon-linear regression, and EC₅₀ values were calculated and shown inTable 11. The reporter assay demonstrate the ability of BCP-84 andBCP-85 to efficiently activate NFAT signal similar to biosimilaratezolizumab.

PD-L1 pathway inhibition by BCP-84 and BCP-85 was studied by determiningthe IL-2 secretion level in mixed lymphocyte reactions (MLR) containingtarget cells expressing PD-L1 (such as dendritic cells), activated Tcells, and BABP. Briefly, human CD4⁺ T cells and allogeneic monocyteswere purified from PBMC using isolation kits (Miltenyl Biotec).Monocytes were induced into dendritic cells. Each well contained 10⁵CD4⁺ T cells and 10⁴ allogeneic dendritic cells with a final workingvolume of 200 μl. Each BABP was added into each well at differentconcentrations. A no-antibody well was used as the background control.Human IgG1 was used as the negative control, and biosimilar atezolizumabwas used as the positive anti-PD-L1 antibody control. After incubatingfor 72 hours at 37° C. in a 5% CO2 incubator, 100 μl medium was takenfrom each testing well for IL-2 measurement (Cisbio).Concentration-dependent secretion of IL-2 in the MLRs was used toextract an EC₅₀ value for BCP-84 and BCP-85 against PD-L1, which iscompared with the EC₅₀ value of backbone antibody atezolizumab (see,Table 12).

CTLA-4 pathway inhibition by the BABPs was studied by determining IL-2secretion level in mixed lymphocyte reactions containing target cellsexpressing CD80, activated T cells, and each BABP. Human CD4⁺ T cellswere purified from PBMC using isolation kits (Miltenyl Biotec). Eachwell contained 10⁵ CD4⁺ T cells and 10⁴ CHO-K1/human CD80 (CHO-K1 stablyexpressing human CD80) with a final working volume of 200 μl. Each BABPwas added into each well at different concentrations. A no-antibody wellwas used as the background control. Human IgG4 was used as the negativecontrol and ipilimumab (e.g., YERVOY®) was used as the positiveanti-CTLA4 antibody control. CTLA4-Fc (GenScript, Z03373-50) was addedinto the system to initiate the reaction. After incubating for 24 hoursat 37° C. in a 5% CO2 incubator, 100 μl medium was taken from eachtesting well for IL-2 measurement (Cisbio). Concentration-dependentsecretion of IL-2 in the CTLA-4 blockade bioassays was used to extractan EC₅₀ value for the BABPs against CTLA-4, which is compared with theEC₅₀ value of control CTLA-4 antibody ipilimumab (e.g., YERVOY®) (see,Table 11).

TABLE 11 In vitro biological assay of exemplary PD-L1/CTLA-4 BABPs.PD-L1 CTLA-4 Construct EC₅₀ (nM) EC₅₀ (nM) BCP-84 2.1 12.8 BCP-75 2.2 9.2 Biosimilar atezolizumab 2 N/A sdAb-2 N/A 17.0 sdAb-3 N/A 13.1ipilimumab (YERVOY ®) N/A 17.6

TABLE 12 Mixed lymphocyte reactions of exemplary PD-L1/CTLA-4 BABPs.Construct IFN-γ EC₅₀ (nM) IL-2 EC₅₀ (nM) BCP-79 1.45 0.56 BCP-80 1.160.58 Biosimilar atezolizumab 0.44 0.67

Example 6: In Vivo Anti-Tumor Efficacy of PD-L1/CTLA-4 BispecificAntigen Binding Proteins

This example describes in vivo experiments assessing the functionalblockade of PD-L1 and CTLA-4 by BCP-84 and BCP-85. Anti-tumor efficacywas evaluated in tumor models developed with human CTLA-4 Knock-in mice.As biosimilar atezolizumab also binds to mouse PD-L1, humanization ofCTLA-4 in mice enabled direct in vivo evaluation of the efficacy ofBCP-84 and BCP-85 BABPs in a mouse tumor xenograft model.

The mouse xenograft models were prepared by implanting tumor cells intoC57BL/6 CTLA-4 KI mice. A murine colon adenocarcinoma cell line MC38stable expression human PD-L1 was used in this assay. MC38-h PD-L1 KIcells (10⁷) were subcutaneously injected in 8-week-old C57BL/6 CTLA-4 KIMice. Tumor size was measured with a caliper, and tumor volume wascalculated by the modified ellipsoid formula: length×(width)/2. Whentumors reached a volume of approximately 90-130 mm³, mice were randomlyassigned to different treatment groups, which were maintained for 2 or 6weeks. The mice were administered vehicle control, anti-PD-L1 antibody(biosimilar atezolizumab), anti-CTLA-4 antibody (sdAb-2-Fc, orsdAb-3-Fc), combination of biosimilar atezolizumab and anti-CTLA-4antibody (sdAb-2-Fc, or sdAb-3-Fc), or BABP (BCP-84 or BCP-85) byintraperitoneal injection. Efficacy of the BABPs was evaluated byassessing inhibition of tumor size and tumor weight.

As shown in FIG. 25 , the combination of biosimilar atezolizumab andanti-CTLA-4 antibody (sdAb-2-Fc, or sdAb-3-Fc) demonstrated higher tumorinhibition efficacy over either monotherapy in the mouse tumor model.Notably, the anti-tumor efficacy of BCP-84 and BCP-85 was comparable asthe combination therapy.

Example 7: Construction, Expression and Biophysical Characterization ofAng2/VEGF Bispecific Antigen Binding Proteins

This example describes the construction and expression of exemplaryAng2/VEGF BABPs. Four constructs were designed and expressed, eachcomprising two polypeptide chains as follows:

Construct 1 (BCP-49): The first polypeptide comprises from theN-terminus to the C terminus: the V_(H)H domain of an anti-VEGF sdAb, apeptide linker (SEQ ID NO: 13), the heavy chain variable domain V_(H) ofLC10 (anti-Ang2 antibody), and heavy chain constant domains of IgG1. Thesecond polypeptide comprises from the N-terminus to the C-terminus: thelight chain variable domain V_(L) of LC10 (anti-Ang2 antibody), andantibody lambda light chain C_(L) domain. BCP-49 has the format of FIG.9 .

Construct 2 (BCP-50): The first polypeptide comprises from theN-terminus to the C terminus: the heavy chain variable domain V_(H) ofLC10 (anti-Ang2 antibody), heavy chain constant domains of IgG1, apeptide linker (SEQ ID NO: 13), and the V_(H)H domain of an anti-VEGFsdAb. The second polypeptide comprises from the N-terminus to theC-terminus: the light chain variable domain V_(L) of LC10 (anti-Ang2antibody), and antibody lambda light chain C_(L) domain. BCP-50 has theformat of FIG. 4 .

Construct 3 (BCP-51): The first polypeptide comprises from theN-terminus to the C terminus: the heavy chain variable domain V_(H) ofLC10 (anti-Ang2 antibody), and heavy chain constant domains of IgG1. Thesecond polypeptide comprises from the N-terminus to the C-terminus: theV_(H)H domain of an anti-VEGF sdAb, a peptide linker (SEQ ID NO: 13),the light chain variable domain V_(L) of LC10 (anti-Ang2 antibody), andantibody lambda light chain C_(L) domain. BCP-51 has the format of FIG.13 .

Construct 4 (BCP-52): The first polypeptide comprises from theN-terminus to the C terminus: the heavy chain variable domain V_(H) ofLC10 (anti-Ang2 antibody), and heavy chain constant domains of IgG1. Thesecond polypeptide comprises from the N-terminus to the C-terminus: thelight chain variable domain V_(L) of LC10 (anti-Ang2 antibody), antibodylambda light chain C_(L) domain, a peptide linker (SEQ ID NO: 13), andthe V_(H)H domain of an anti-VEGF sdAb. BCP-52 has the format of FIG. 11.

The plasmids of the four BABPs were prepared and transiently expressedin CHO cells. The BABPs were purified by one-step protein Achromatography and store in 4% Sucrose, 50 mM Histidine, 50 mM Arginine,pH 6.0 buffer. The composition and purity of the purified BABPs wereanalyzed by SDS-PAGE under both reduced and non-reduced conditions. Thesizes of the chains as well as the full-length protein of BABP moleculesare consistent with their calculated molecular mass based on the aminoacid sequences. To further study the physical properties of the fourBABPs in solution, size exclusion chromatography was used to analyzeeach protein. All four proteins exhibited a single major peak,demonstrating physical homogeneity as monomeric BABP molecules. Asummary of this data is shown in the Table 13.

TABLE 13 Production of exemplary Ang2/VEGF BABPs. Host cell ExpressionMonomeric BABP line (mg/L) molecule Buffer BCP-49 CHO-3E7 90.3 97.92% 4%Sucrose, 50 BCP-50 97.5 98.20% mM Histidine, BCP-51 67.5 98.16% 50 mMArginine, BCP-52 95.5 97.77% pH 6.0

Example 8: In Vitro Functional Assays of Ang2/VEGF Bispecific AntigenBinding Proteins

The binding kinetics of BABPs to rhAng2 and rhVEGF was determined bySurface Plasmon Resonance with a BIACORE® T200 instrument using HBS-EP(10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.05% Tween-20).Briefly, goat anti-human IgG polyclonal antibody was directlyimmobilized across a CMS biosensor chip using a standard ammine couplingkit according to manufacturer's instructions. Purified FIT-Ig sampleswere diluted in HEPES-buffered saline for capture across goat anti-humanIgG Fc specific reaction surfaces, and injected over reaction matricesat a flow rate of 5 μl/min. The association and dissociation rateconstants, k_(on) and k_(off) were determined under a continuous flowrate of 30 μl/min. The kinetics data is shown in Table 14. The antibodyaffinities of the Ang2/VEGF BABPs are similar to the corresponding4-chain antibody LC10, or anti-VEGF sdAbs fused to an Fc fragment.

TABLE 14 Binding data of exemplary Ang2/VEGF BABPs. VEGF Ang2 ConstructKD (nM) EC50 KD (nM) EC50 BCP-49 0.51 0.25 2.3 3.1 BCP-50 0.48 0.39 8.83.9 BCP-51 0.32 0.32 1.9 3.5 BCP-52 1.29 0.36 1.4 3.8 sdAbVEGF-Fc 0.350.23 LC10 3.4 2.8

To assess the bioactivity of Ang2/VEGF BABPs targeting VEGF, HUVEC cellswere used for a mitogenic assay. HUVEC cells were seeded in 6-wellplates at a density of 6×10³ cells per well, and cultured in low glucoseDulbecco's modified Eagle's medium (DMEM) (GIBCO) supplemented with 10%calf serum, 2 mM glutamine, and antibiotics (growth medium). Anti-VEGFsdAb fused to an Fc fragment (“sdAbVEGF-Fc”) was then added atconcentrations ranging between 1 and 5000 ng/ml. After 2-3 hours,purified rhVEGF165 was added at a final concentration of 3 ng/ml. Afterfive or six days, cells were dissociated by exposure to trypsin andcounted in a Coulter counter. Variation from the mean number of cellsdid not exceed 10%. Data were analyzed by a four-parameter curve fittingprogram. As shown in Table 14, all four Ang2/VEGF BABPs have comparablebiological activities targeting VEGF as sdAbVEGF-Fc.

To assess Ang-2 inhibition by the BABPs, Tie2 phosphorylation, which wasinduced by inhibition of Ang-2, was measured as follows. HEK293-Tie2cells were stimulated with Ang-2 for 5 minutes in the presence orabsence of LC10 antibody or each BABP. Then, levels of phosphorylatedTie2 (“P-Tie2”) in cell lysates were quantified using a sandwich ELISAaccording to the manufacturer's instructions. IC₅₀ values weredetermined using GraphPad PRISM™ version 6. As shown in Table 14, allfour Ang2/VEGF BABPs have comparable biological activity targeting Ang2as LC10.

Example 9: In Vivo Efficacy of Ang2/VEGF Bispecific Antigen BindingProteins

A375 xenografts were used to evaluate the anti-tumor efficacies ofAng2/VEGF BABPs described in Examples 7-8 as compared to anti-Ang2 sdAband anti-VEGF antibody monotherapy or combination therapy.

10⁷ A375 cells were subcutaneously injected to 6-week-old Balb/c nudemice. Tumor size was measured with a caliper, and tumor volume wascalculated by the modified ellipsoid formula: length×(width)/2. Whentumors reached a volume of approximately 90-130 mm³, mice were randomlyassigned to different treatment groups, which were maintained for 2 or 6weeks. The mice were administered vehicle control, LC10, sdAbVEGF-Fc,LC10+sdAbVEGF-Fc combination, or BCP-49 intravenously twice a week.

Tumor volume was measured twice a week and data is shown in FIG. 26A.Compared to the vehicle control, significant inhibition of tumor growthwas observed in the sdAbVEGF-Fc, LC10+sdAbVEGF-Fc combination therapy,and BCP-49 treatment groups. Notably, synergic activity was observed inthe BCP49-treated group compared to the combination therapy group.

Tumor weight was also measured after study completion. Consistent withthe tumor volume results, BCP49 was more effective than theLC10+sdAbVEGF-Fc combination therapy in reducing tumor weight, as shownin FIG. 26B.

All citations throughout the disclosure are hereby expresslyincorporated by reference.

What is claimed is:
 1. A multispecific antigen binding protein (MABP)comprising: (a) a first antigen binding portion comprising a heavy chainvariable domain (V_(H)) and a light chain variable domain (V_(L)),wherein the V_(H) and V_(L) together form an antigen-binding site thatspecifically binds a first epitope, and (b) a second antigen bindingportion comprising a single-domain antibody (sdAb) that specificallybinds a second epitope, and (c) an Fc region, wherein the first antigenbinding portion and the second antigen binding portion are fused to eachother, and wherein the first antigen binding portion is connected to theFc region via the second antigen binding portion. 2-3. (canceled)
 4. TheMABP of claim 1, wherein the MABP is bispecific.
 5. The MABP of claim 1,wherein: (i) the first antigen binding portion is a full-length antibodyconsisting of two heavy chains and two light chains; or (ii) the firstantigen binding portion is an antibody fragment comprising a heavy chaincomprising the V_(H) and a light chain comprising the V_(L). 6-11.(canceled)
 12. The MABP of claim 1, wherein the second antigen bindingportion is a Fab-like domain comprising: (i) a first polypeptide chaincomprising a sdAb fused to a C_(H)1 domain; or (ii) a first polypeptidechain comprising a sdAb fused to a C_(H)1 domain, and a secondpolypeptide chain comprising a second single-domain antibody fused to aC_(L) domain. 13-17. (canceled)
 18. The MABP of claim 1, wherein thefirst antigen binding portion and the second antigen binding portion arefused to each other via a peptide bond or a peptide linker.
 19. The MABPof claim 18, wherein the peptide linker is no more than about 30 aminoacids long.
 20. The MABP of claim 19, wherein the peptide linkercomprises the amino acid sequence of SEQ ID NO: 1, or
 13. 21-22.(canceled)
 23. The MABP of claim 1, wherein: (i) the first epitope isfrom an immune checkpoint molecule; (ii) the second epitope is from animmune checkpoint molecule; or (iii) the first epitope and the secondepitope are from an immune checkpoint molecule.
 24. The MABP of claim23, wherein the immune checkpoint molecule is selected from the groupconsisting of PD-1, PD-L1, PD-L2, CTLA-4, B7-H3, TIM-3, LAG-3, VISTA,ICOS, 4-1BB, OX40, GITR, and CD40.
 25. The MABP of claim 24, wherein thefirst antigen binding portion is an anti-PD-1 antibody or antigenbinding fragment thereof or an anti-PD-L1 antibody or antigen bindingfragment thereof. 26-30. (canceled)
 31. The MABP of claim 30, whereinthe second antigen binding portion comprises an anti-CTLA-4 sdAb. 32.The MABP of claim 1, wherein the first epitope is from a tumor antigenor a pro-inflammatory molecule.
 33. The MABP of claim 32, wherein: (i)the tumor antigen is selected from the group consisting of HER2, BRAF,EGFR, VEGFR2, CD20, RANKL, CD38, and CD52; or (ii) the pro-inflammatorymolecule is selected from the group consisting of IL-1β, TNF-α, IL-5,IL-6, IL-6R, and eotaxin-1. 34-36. (canceled)
 37. The MABP of claim 1,wherein: (i) the first antigen binding portion is an anti-Ang2 antibodyor antigen binding fragment thereof; and/or (ii) the second antigenbinding portion is an anti-VEGF sdAb. 38-51. (canceled)
 52. Apharmaceutical composition comprising the MABP of claim 1 and apharmaceutically acceptable carrier.
 53. A method of treating a diseasein an individual, comprising administering to the individual aneffective amount of the pharmaceutical composition of claim
 52. 54-57.(canceled)
 58. The MABP of claim 1, wherein: (i) the first antigenbinding portion comprises a Fab, wherein the N-terminus of the sdAb isfused to the C-terminus of the Fab, and the C-terminus of the sdAb isfused to the N-terminus of the Fc region; (ii) (a) the first antigenbinding portion is a Fab; (b) the second antigen binding portion is aFab-like domain comprising a sdAb, wherein the N-termini of the Fab-likedomain is fused to the C-termini of the Fab, and the C-termini of theFab-like domain is fused to the N-terminus of the Fc region; or (iii)(a) the first antigen binding portion is a scFv; (b) the second antigenbinding portion is a Fab-like domain comprising a sdAb, wherein theN-termini of the Fab-like domain is fused to the C-terminus of the scFv,and the C-termini of the Fab-like domain is fused to an N-terminus ofthe Fc region.
 59. The MABP of claim 1, comprising two identical firstpolypeptides and two identical second polypeptides, wherein: (i) (a) thetwo identical first polypeptides each comprise, from the N-terminus tothe C-terminus: V_(H)-C_(H)1-an optional peptidelinker-sdAb-C_(H)2-C_(H)3; and (b) the two identical second polypeptideseach comprise a V_(L)-C_(L); (ii) (a) the two identical firstpolypeptides each comprise, from the N-terminus to the C-terminus:V_(H)-C_(H)1-an optional peptide linker-sdAb-C_(H)1-C_(H)2-C_(H)3; and(b) the two identical second polypeptides each comprise, from theN-terminus to the C-terminus: V_(L)-C_(L)-an optional peptidelinker-sdAb-C_(L); or, (iii) (a) the two identical first polypeptideseach comprise, from the N-terminus to the C-terminus: scFv-an optionalpeptide linker-sdAb-C_(H)1-C_(H)2-C_(H)3; and (b) the two identicalsecond polypeptides each comprise, from the N-terminus to theC-terminus: sdAb-C_(L).
 60. The MABP of claim 1, comprising fourpolypeptide chains with structures from the N-terminus to the C-terminusas follows: (i) (1) V_(L)-C_(L); (2) V_(H)-C_(H)1-V_(H)H-C_(H)2-C_(H)3;(3) V_(H)-C_(H)1-V_(H)H-C_(H)2-C_(H)3; and (4) V_(L)-C_(L), whereinV_(H) and V_(L) of polypeptide chains (1) and (2) forms an antigenbinding site that specifically binds a first copy of the first epitope,V_(H) and V_(L) of polypeptide chains (3) and (4) forms an antigenbinding site that specifically binds a second copy of the first epitope,and each V_(H)H specifically binds a copy of the second epitope; (ii)(1) V_(L)-C_(L)-V_(H)H-C_(L); (2)V_(H)-C_(H)1-V_(H)H-C_(H)1-C_(H)2-C_(H)3; (3)V_(H)-C_(H)1-V_(H)H-C_(H)1-C_(H)2-C_(H)3; and (4)V_(L)-C_(L)-V_(H)H-C_(L), wherein V_(H) and V_(L) of polypeptide chains(1) and (2) forms an antigen binding site that specifically binds afirst copy of the first epitope, V_(H) and V_(L) of polypeptide chains(3) and (4) forms an antigen binding site that specifically binds asecond copy of the first epitope, and each V_(H)H specifically binds acopy of the second epitope; or (iii) (1) V_(H)H-C_(L); (2)V_(L)-V_(H)-V_(H)H-C_(H)1-C_(H)2-C_(H)3; (3)V_(L)-V_(H)-V_(H)H-C_(H)1-C_(H)2-C_(H)3; and (4) V_(H)H-C_(L), whereinV_(H) and V_(L) of polypeptide chains (2) and (3) each forms an scFvthat specifically binds a copy of the first epitope, and each V_(H)Hspecifically binds a copy of the second epitope.
 61. The method of claim53, wherein the disease is cancer, an inflammatory disease, or anautoimmune disease.